Source: AGRICULTURAL RESEARCH SERVICE submitted to
CHARACTERIZATION OF EXPRESSION PATTERNS OF STRESS TOLERANCE GENES IN WHEAT
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
USDA INHOUSE
Reporting Frequency
Annual
Accession No.
0407369
Grant No.
(N/A)
Project No.
5348-21430-003-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jun 18, 2003
Project End Date
Apr 13, 2008
Grant Year
(N/A)
Project Director
SKINNER D Z
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
PULLMAN,WA 99164
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20315431080100%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1543 - Soft white wheat;

Field Of Science
1080 - Genetics;
Goals / Objectives
Determine biological and molecular processes that improve stress tolerance in wheat, primarily cold stress. The specific objectives are: Characterize expression level changes of genes from known metabolic pathways in wheat plants exposed to stress factors, primarily cold stress. Use a wheat diallel across to characterize the genetic control of the accumulation of metabolic compounds shown to accumulate to higher levels in response to cold exposure, and genetic control of physiological changes associated with response to temperature, such as rates of acclimation and deacclimation. Develop wheat cell lines or whole plants with artificially altered levels of metabolites shown to be correlated with stress tolerance in other plant systems, and test for induction of stress tolerance.
Project Methods
Use microarrays to analyze the expression levels of genes in the known metabolic pathways of wheat plants as they react to cold stress. Use a wheat diallel cross to characterize the genetic control of the accumulation of metabolic compounds shown to accumulate to higher levels in response to cold exposure, and genetic control of physiological changes associated with response to temperature. Develop wheat cell lines with artificially altered levels of metabolites shown to be correlated with stress tolerance in other plant systems, and test for induction of stress tolerance. BL-I; 12/16/2002. Replaces 5348-21430-002-00D (6/03).

Progress 06/18/03 to 04/13/08

Outputs
Progress Report Objectives (from AD-416) Determine biological and molecular processes that improve stress tolerance in wheat, primarily cold stress. The specific objectives are: Characterize expression level changes of genes from known metabolic pathways in wheat plants exposed to stress factors, primarily cold stress. Use a wheat diallel across to characterize the genetic control of the accumulation of metabolic compounds shown to accumulate to higher levels in response to cold exposure, and genetic control of physiological changes associated with response to temperature, such as rates of acclimation and deacclimation. Develop wheat cell lines or whole plants with artificially altered levels of metabolites shown to be correlated with stress tolerance in other plant systems, and test for induction of stress tolerance. Approach (from AD-416) Use microarrays to analyze the expression levels of genes in the known metabolic pathways of wheat plants as they react to cold stress. Use a wheat diallel cross to characterize the genetic control of the accumulation of metabolic compounds shown to accumulate to higher levels in response to cold exposure, and genetic control of physiological changes associated with response to temperature. Develop wheat cell lines with artificially altered levels of metabolites shown to be correlated with stress tolerance in other plant systems, and test for induction of stress tolerance. Replaces 5348-21430-002-00D (6/03). Significant Activities that Support Special Target Populations A study of freezing tolerance in 20 segregating populations was completed. This study revealed that freezing tolerance, in the populations studied, is generally conditioned by genes that have a degree of dominance, however, one wheat line had a strongly dominant gene that condition freezing sensitivity. A study characterizing this gene was published in Plant Breeding in 2008. These results provide new information on the genes involved in the response of wheat to freezing stress, thereby providing guidance to breeding efforts to improve cold tolerance and winter survival. The wheat germplasm in which the unique, dominant, cold sensitivity gene was discovered was released in 2008 through the USDA-ARS system. Two lines with apparently improved cold tolerance, relative to the most tolerant parent, were identified from the study of segregating populations. A description of those lines has been submitted to a journal for publication. A study of the ability of 26 wheat lines to withstand 15 and 20 weeks frozen at -5C was completed and has been accepted for publication by the Canadian Journal of Plant Science. The LT50, the temperature predicted to kill 50% of the plants, was significantly related to survival ability at both time points, but some wheat lines survived better than predicted by the LT50. The levels of expression of thousands of genes in wheat plants undergoing freezing stress were assayed using microarrays. Subsets of these genes were shown to respond to severe freezing stress, but not necessarily to less severe stress. This result suggested that different freezing tolerance mechanisms in wheat respond to different levels of stress. This research addresses National Program 301 Action Plan Component 3. Genetic Improvement of Crops; Problem Statement 3C: Germplasm Enhancement/Release of Improved Genetic Resources and Varieties.

Impacts
(N/A)

Publications


    Progress 10/01/06 to 09/30/07

    Outputs
    Progress Report Objectives (from AD-416) Determine biological and molecular processes that improve stress tolerance in wheat, primarily cold stress. The specific objectives are: Characterize expression level changes of genes from known metabolic pathways in wheat plants exposed to stress factors, primarily cold stress. Use a wheat diallel across to characterize the genetic control of the accumulation of metabolic compounds shown to accumulate to higher levels in response to cold exposure, and genetic control of physiological changes associated with response to temperature, such as rates of acclimation and deacclimation. Develop wheat cell lines or whole plants with artificially altered levels of metabolites shown to be correlated with stress tolerance in other plant systems, and test for induction of stress tolerance. Approach (from AD-416) Use microarrays to analyze the expression levels of genes in the known metabolic pathways of wheat plants as they react to cold stress. Use a wheat diallel cross to characterize the genetic control of the accumulation of metabolic compounds shown to accumulate to higher levels in response to cold exposure, and genetic control of physiological changes associated with response to temperature. Develop wheat cell lines with artificially altered levels of metabolites shown to be correlated with stress tolerance in other plant systems, and test for induction of stress tolerance. BL-I; 12/16/2002. Replaces 5348-21430-002- 00D (6/03). Accomplishments Method of distinguishing freezing tolerance mechanisms described. Numerous mechanisms are known to be involved in the response of plants to cold temperature, but identifying plant lines in which the relative significance of mechanisms differ has not been accomplished. ARS researchers in Pullman, WA used a unique method of quantifying plants' response to components of the freezing process. Wheat lines that appear to use predominantly different mechanisms in cold response were identified. This method holds promise as a means to identify parental lines to use in the improvement of cold tolerance. This work was done in the Wheat Genetics, Quality, Physiology, and Disease Research Unit, Pullman, WA, and addresses NP301 Component 3. Genetic Improvement of Crops, Problem Statement 3A: Genetic Theory and Methods of Crop Improvement. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 3

    Impacts
    (N/A)

    Publications


      Progress 10/01/05 to 09/30/06

      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? Abiotic stress is a major impediment to wheat productivity. In the Pacific Northwest, cold temperature is a very significant abiotic stress to both winter and spring wheat. The problem is being addressed through systematically examining the responses to cold of metabolic and regulatory pathways in wheat. Wheat is a very significant source of food for the US and the world. Each year, millions of kilograms of potential grain yield fails to materialize because of stress imposed on the plants by cold temperatures in the fall, winter and spring months. In some years, some fields are not harvested because of severe winter injury reducing the yield to very low levels. Intolerance of cold temperatures also limits the geographic range in which wheat varieties can be grown. A single additional degree of cold tolerance may significantly extend the range of productive growth of a wheat variety. The project has three specific goals: Characterize expression level changes of genes from known metabolic pathways in wheat plants exposed to stress factors, primarily cold stress; use a wheat diallel cross to characterize the genetic control of the accumulation of metabolic compounds shown to accumulate to higher levels in response to cold exposure, and genetic control of physiological changes associated with response to temperature, such as rates of acclimation and deacclimation; develop wheat cell lines or whole plants with artificially altered levels of metabolites shown to be correlated with stress tolerance in other plant systems, and test for induction of stress tolerance. The research to be undertaken falls under National Program 301 - Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement, and contributes to component 2, Genomic Characterization and Genetic Improvement. Successful outcomes from this project will contribute specifically to the three stated goals of component 2: "Sources of useful and important genes should be identified and incorporated into crop germplasm. The genetic bases of crop, microbial and beneficial insect genepools should be broadened through genetic improvement, to combat potential losses due to pests, diseases, and stresses imposed by the natural environment. The underlying genetic bases of key agronomic/horticultural traits should be elucidated, because that information may greatly facilitate the deployment of such traits in improved germplasm." 2. List by year the currently approved milestones (indicators of research progress) Year 1 (June 2003- June 2004) Define genes to (micro)array, design oligos, construct arrays, preliminary hybridizations. Select parents, make initial diallel crosses. Establish cell culture lines from wheat cultivars known to differ in cold hardiness, become familiar with LT50 scoring on cell cultures. Year 2 (June 2004- June 2005) Hybridization (of microarrays) with RNA from time-course exposed plants, QRT-PCR to confirm. Grow and vernalize F1s (of diallel). Do chemical analyses. Produce F2 seed in greenhouse. Develop procedures for treating cells with specific chemicals, (concentration and length of exposure, etc. ) then testing LT50. Year 3 (June 2005- June 2006) Hybridization/QRT-PCR with RNA from a range of wheat lines identification of lines differing in response. Grow F2 seed, analyze acclimation and deacclimation rates, make selections based on all traits. Test range of chemicals with known metabolite induction properties, do chemical analyses, measure LT50. Begin transformation work if warranted. Year 4 (June 2006- June 2007) Crossing and cold tolerance testing (F1) of lines identified in previous step. Grow selected F2 lines in the field for F3 seed, evaluate field cold- tolerance make selections based on all traits. Combine results of Objectives 1 and 2 with prior results of this objective. Use cultured cells from derived lines to test combinations of increased metabolites on LT50. Year 5 (June 2007- June 2008) Inbred line development, cold testing, QRT-PCR analysis. Test selected lines for LT50, make further selections based on traits experience thus far shows to be useful, combine with plant lines from Objective 1 for further development. Use (cell culture) results of previous 4 years to guide plant selections for further improvement. Complete transformation studies, if warranted. 4a List the single most significant research accomplishment during FY 2006. Possible major gene regulating cold tolerance identified. The specific genes involved in cold temperature response of wheat remain poorly defined. We used unique wheat lines to investigate the inheritance of response to harsh cold temperature. An apparent dominant major gene conditioning freezing sensitivity was identified. This gene holds promise as a significant tool to be used in defining fundamental processes involved in plant death due to cold temperature. This research addresses NP301 action plan Component 3: Genetic Improvement of Crops; Problem Statement 3A: Genetic Theory and Methods of Crop Improvement. 4b List other significant research accomplishment(s), if any. Freezing tolerance components investigated. Differences among wheat lines in mechanisms involved in response to cold temperature have not been described. We used regression component analysis to investigate tolerance of phases of the freezing process in six wheat cultivars. Significant differences were found in relative tolerance of various phases. These results provide clues for finding wheat lines that may have significantly different mechanisms of coping with freezing temperatures, which may be exploitable for cold tolerance improvement in cultivated wheat. This research addresses NP301 action plan Component 3: Genetic Improvement of Crops; Problem Statement 3A: Genetic Theory and Methods of Crop Improvement. 5. Describe the major accomplishments to date and their predicted or actual impact. This research addresses NP301 action plan Component 3: Genetic Improvement of Crops; Problem Statement 3A: Genetic Theory and Methods of Crop Improvement. Identified a pattern of inheritance of freezing tolerance indicative of a dominant gene conditioning freezing sensitivity. This finding is important because it provides a tool to investigate the fundamental processes involved in the death of wheat plants due to freezing. Characterized a genomic copy of the wheat manganese superoxide dismutase gene including regions involved in the control of expression and quantified the stability of the expressed form of this gene. This information provides new information on the control of expression of one of the genes involved in response to abiotic stress, providing information on the regulation of this gene broadening the knowledge of gene regulation in stress response. Identified previously unknown diversity within an antioxidant gene family in wheat and demonstrated that some members of this gene family are much more responsive to cold temperature than others. This information provides heretofore-unknown targets for molecular breeding to enhance the ability of wheat plants to respond to cold stress. Identified previously unknown patterns of response of phospholipid accumulation during cold acclimation, suggestive of phospholipid signaling. This information provides the basis for investigation of novel signal cascades involved in response to cold temperature. Identified several genes that are responsive to cold, heat, bacterial colonization, or combinations of these stress factors. The information generated by these accomplishments is used by wheat scientists to work towards improvement of stress tolerance of wheat. The fundamental knowledge gained by these accomplishments provides avenues of research that were unknown prior to their discovery. The potential impact of these results lies in the further definition of the principals described and their application to plant improvement. 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? During FY06, antioxidant gene structure and expression information and inheritance data associated with cold acclimation in wheat was made available to the scientific community through publication and presentations at professional meetings. Wheat germplasms lines developed through this project are expected to become available within four years. Transfer to the end user will be through the conventional plant breeding process, typically requiring several years to complete. 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). Presentation to the Crop Science Society of America Annual Meeting: Distributions of cold tolerance in F3 wheat populations. Nov 8, 2006. Salt Lake City, UT.

      Impacts
      (N/A)

      Publications

      • Baek, K., Skinner, D.Z., Ling, P., Chen, X. 2006. Molecular structure and organization of the wheat genomic manganese superoxide dismutase gene. Genome 49:209-218.
      • Baek, K., Skinner, D.Z. 2005. Differential mrna stability to endogenous ribonucleases of the coding region and 3' untranslated regions of wheat (triticum aestivum l.) manganese superoxide dismutase genes. Plant Cell Reports. 25:133-139.
      • Baek, K., Skinner, D.Z. 2005. Differential expression of manganese superoxide dismutase sequence variants in near isogenic lines of wheat during cold acclimation. Plant Cell Reports. 25:223-230.
      • Skinner, D.Z. 2006. Plant abiotic stress. Environmental Quality 35:955A.
      • Skinner, D.Z. 2005. Genetically modified planet. J. Envir. Qual. 34:2335- 2336.
      • Skinner, D.Z., Garland Campbell, K.A. 2005. Distributions of cold tolerance in f3 wheat populations. ASA-CSSA-SSSA Annual Meeting Abstracts, Nov 6-10, Salt Lake City, UT. 240:15.


      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? Abiotic stress is a major impediment to wheat productivity. In the Pacific Northwest, cold temperature is a very significant abiotic stress to both winter and spring wheat. The problem is being addressed through systematically examining the responses to cold of metabolic and regulatory pathways in wheat. Wheat is a very significant source of food for the US and the world. Each year, millions of kilograms of potential grain yield fails to materialize because of stress imposed on the plants by cold temperatures in the fall, winter and spring months. In some years, some fields are not harvested because of severe winter injury reducing the yield to very low levels. Intolerance of cold temperatures also limits the geographic range in which wheat varieties can be grown. A single additional degree of cold tolerance may significantly extend the range of productive growth of a wheat variety. The project has three specific goals: Characterize expression level changes of genes from known metabolic pathways in wheat plants exposed to stress factors, primarily cold stress; use a wheat diallel cross to characterize the genetic control of the accumulation of metabolic compounds shown to accumulate to higher levels in response to cold exposure, and genetic control of physiological changes associated with response to temperature, such as rates of acclimation and deacclimation; develop wheat cell lines or whole plants with artificially altered levels of metabolites shown to be correlated with stress tolerance in other plant systems, and test for induction of stress tolerance. The research to be undertaken falls under National Program 301 - Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement, and contributes to component 2, Genomic Characterization and Genetic Improvement. Successful outcomes from this project will contribute specifically to the three stated goals of component 2: Sources of useful and important genes should be identified and incorporated into crop germplasm. The genetic bases of crop, microbial and beneficial insect genepools should be broadened through genetic improvement, to combat potential losses due to pests, diseases, and stresses imposed by the natural environment. The underlying genetic bases of key agronomic/horticultural traits should be elucidated, because that information may greatly facilitate the deployment of such traits in improved germplasm. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (June 2003 - June 2004) Define genes to (micro)array, design oligos, construct arrays, preliminary hybridizations. Select parents, make initial diallel crosses. Establish cell culture lines from wheat cultivars known to differ in cold hardiness, become familiar with LT50 scoring on cell cultures. Year 2 (June 2004 - June 2005) Hybridization (of microarrays) with RNA from time-course exposed plants, QRT-PCR to confirm. Grow and vernalize F1s (of diallel). Do chemical analyses. Produce F2 seed in greenhouse. Develop procedures for treating cells with specific chemicals, (concentration and length of exposure, etc.) then testing LT50. Year 3 (June 2005- June 2006) Hybridization/QRT-PCR with RNA from a range of wheat lines identification of lines differing in response. Grow F2 seed, analyze acclimation and deacclimation rates, make selections based on all traits. Test range of chemicals with known metabolite induction properties, do chemical analyses, measure LT50. Begin transformation work if warranted. Year 4 (June 2006- June 2007) Crossing and cold tolerance testing (F1) of lines identified in previous step. Grow selected F2 lines in the field for F3 seed, evaluate field cold- tolerance make selections based on all traits. Combine results of Objectives 1 and 2 with prior results of this objective. Use cultured cells from derived lines to test combinations of increased metabolites on LT50. Year 5 (June 2007- June 2008) Inbred line development, cold testing, QRT-PCR analysis. Test selected lines for LT50, make further selections based on traits experience thus far shows to be useful, combine with plant lines from Objective 1 for further development. Use (cell culture) results of previous 4 years to guide plant selections for further improvement. Complete transformation studies, if warranted. 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. Hybridization (of microarrays) with RNA from time-course exposed plants, QRT-PCR to confirm. Milestone Fully Met 2. Grow and vernalize F1s (of diallel). Do chemical analyses. Produce F2 seed in greenhouse. Milestone Fully Met 3. Develop procedures for treating cells with specific chemicals, (concentration and length of exposure, etc.) then testing LT50. Milestone Fully Met 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? Year 3 (June 2005- June 2006) (1) Hybridization/QRT-PCR with RNA from a range of wheat lines; identification of lines differing in response. By year three we will have identified genes differentially responsive in wheat lines differing in cold tolerance. We will use QRT - PCR to identify specific wheat lines strongly differing in regulation of these genes. These lines will then be used to quantifying the impact of these individual genes on cold tolerance. (2) Grow F2 seed, analyze acclimation and deacclimation rates, make selections based on all traits. Seed for this study will be produced in the field in 2004 - 2005. Acclimation and deacclimation rates will be evaluated in controlled environment chambers. We currently are working to develop protocols for deacclimation rate studies. (3) Test range of chemicals with known metabolite induction properties, do chemical analyses, measure LT50. Work will center on the identification of specific metabolites induced, and genes concomitantly induced, in response to cold treatment and treatment with choline chloride and glycine betaine, or other suitable chemicals we identify. Year 4 (June 2006- June 2007) (1) Crossing and cold tolerance testing (F1) of lines identified in previous step. This phase of the project will concentrate on testing hypotheses and predictions developed from the previous milestones. Plants characterized for specific metabolite accumulation and cold tolerance will be crossed and the predicted cold tolerance will be tested. (2) Grow selected F2 lines in the field for F3 seed, evaluate field cold- tolerance make selections based on all traits. These F2 lines will be grown from plants with known cold tolerance and the F2 plants will have been selected to represent the range of cold tolerance and metabolite accumulation found in previous project milestones. (3) Combine results of Objectives 1 and 2 with prior results of this objective. Use cultured cells from derived lines to test combinations of increased metabolites on LT50. Year 5 (June 2007- June 2008) Inbred line development, cold testing, QRT-PCR analysis. Test selected lines for LT50, make further selections based on traits experience thus far shows to be useful, combine with plant lines from Objective 1 for further development. Use (cell culture) results of previous 4 years to guide plant selections for further improvement. Complete transformation studies, if warranted. These results will expand our knowledge of the processes wheat plants undergo when exposed to cold temperatures, and provide specific gene to target in molecular breeding approaches to improved cold temperature response. 4a What was the single most significant accomplishment this past year? The control of expression of genes involved in response to stresses involves physical components of the genes. We sequenced and thoroughly characterized a wheat manganese superoxide dismutase gene, an important component of response to cold temperature. Specific physical motifs involved in the control of expression of this gene were found. This accomplishment is important because it provides a new avenue of investigation into the control of response of wheat plants to cold temperature. 4b List other significant accomplishments, if any. The inheritance of response to cold temperature in wheat is poorly understood. We investigated the level of cold tolerance in F3 populations of unrelated parent lines with similar cold tolerance. A bimodal distribution of survival of exposure to damaging temperatures was found, suggesting a complex but predictable genetic control mechanism. This accomplishment is important because it provides materials and information leading to a greater understanding of the genetic control of stress tolerance in wheat. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Characterized a genomic copy of the wheat manganese superoxide dismutase gene including regions involved in the control of expression and quantified the stability of the expressed form of this gene. This information provides new information on the control of expression of one of the genes involved in response to abiotic stress, providing information on the regulation of this gene broadening the knowledge of gene regulation in stress response. Identified previously unknown diversity within an antioxidant gene family in wheat and demonstrated that some members of this gene family are much more responsive to cold temperature than others. This information provides heretofore-unknown targets for molecular breeding to enhance the ability of wheat plants to respond to cold stress. Identified previously unknown patterns of response of phospholipid accumulation during cold acclimation, suggestive of phospholipid signaling. This information provides the basis for investigation of novel signal cascades involved in response to cold temperature. 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? A method for efficient use of microarrays with wheat has been made available to the research community through publication. Phospholipid production data associated with cold acclimation in wheat has been made available to the scientific community through presentations at professional meetings. Wheat germplasms lines developed through this project are expected to become available within five years. Transfer to the end user will be through the conventional plant breeding process, typically requiring several years to complete.

      Impacts
      (N/A)

      Publications

      • Skinner, D.Z., Muthukrishnan, S., Liang, G.H. 2004. In: Transformation, a powerful tool for crop improvement. In: Skinner, D.Z, Liang, G.H., editors. Genetically Modified Crops . Binghamton, NY: Haworth Press. p. 1-16.
      • Skinner, D.Z., Okubara, P.A., Hyun-Baek, K., Call, D.R. 2005. Long oligonucleotide microarrays in wheat: evaluation of hybridization signal amplification and an oligonucleotide-design computer script. Functional and Integrative Genomics. Vol 5, Number 2, pgs 70-79.
      • Baek, K.-H. and Skinner, D.Z. 2004. Quantitative real-time pcr method to detect changes in specific transcript and total RNA amounts. Electronic Journal of Biotechnology. ISSN0717-3458. Vol. 7:1:55-60.
      • Kisha, T.J., Johnson, R.C., Skinner, D.Z., Bauchan, G.R., Greene, S.L. 2004. Variance of molecular genetic distances among alfalfa synthetic populations by marker type. Agronomy Abstracts. #5781.
      • Bellinger, B.S., Skinner, D.Z., Halls, S.C., Garland Campbell, K.A., Siems, W. 2004. Phospholipid dynamics in wheat leaves during cold acclimation. Agronomy Abstracts. #4690.
      • Yi, S., Liang, A., Wang, J., Skinner, D.Z. 2003. A phylogeny study of medicago species based on ribosomal dna its sequences. Acta Botanica Sinica 23(2):242-246.


      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? Abiotic stress is a major impediment to wheat productivity. In the Pacific Northwest, cold temperature is a very significant abiotic stress to both winter and spring wheat. The problem is being addressed through systematically examining the responses to cold of metabolic and regulatory pathways in wheat. Wheat is a very significant source of food for the US and the world. Each year, millions of kilograms of potential grain yield fails to materialize because of stress imposed on the plants by cold temperatures in the fall, winter and spring months. In some years, some fields are not harvested because of severe winter injury reducing the yield to very low levels. Intolerance of cold temperatures also limits the geographic range in which wheat varieties can be grown. A single additional degree of cold tolerance may significantly extend the range of productive growth of a wheat variety. The project has three specific goals: Characterize expression level changes of genes from known metabolic pathways in wheat plants exposed to stress factors, primarily cold stress; use a wheat diallel cross to characterize the genetic control of the accumulation of metabolic compounds shown to accumulate to higher levels in response to cold exposure, and genetic control of physiological changes associated with response to temperature, such as rates of acclimation and deacclimation; develop wheat cell lines or whole plants with artificially altered levels of metabolites shown to be correlated with stress tolerance in other plant systems, and test for induction of stress tolerance. The research to be undertaken falls under National Program 301 - Plant, Microbial, and Insect Genetic Resources, Genomics and Genetic Improvement, and contributes to component 2, Genomic Characterization and Genetic Improvement. Successful outcomes from this project will contribute specifically to the three stated goals of component 2: Sources of useful and important genes should be identified and incorporated into crop germplasm. The genetic bases of crop, microbial and beneficial insect genepools should be broadened through genetic improvement, to combat potential losses due to pests, diseases, and stresses imposed by the natural environment. The underlying genetic bases of key agronomic/horticultural traits should be elucidated, because that information may greatly facilitate the deployment of such traits in improved germplasm. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (June 2003- June 2004) Define genes to (micro)array, design oligos, construct arrays, preliminary hybridizations. Select parents, make initial diallel crosses. Establish cell culture lines from wheat cultivars known to differ in cold hardiness, become familiar with LT50 scoring on cell cultures. Year 2 (June 2004- June 2005) Hybridization (of microarrays) with RNA from time-course exposed plants, QRT-PCR to confirm. Grow and vernalize F1s (of diallel). Do chemical analyses. Produce F2 seed in greenhouse. Develop procedures for treating cells with specific chemicals, (concentration and length of exposure, etc. ) then testing LT50. Year 3 (June 2005- June 2006) Hybridization/QRT-PCR with RNA from a range of wheat lines identification of lines differing in response. Grow F2 seed, analyze acclimation and deacclimation rates, make selections based on all traits. Test range of chemicals with known metabolite induction properties, do chemical analyses, measure LT50. Begin transformation work if warranted. Year 4 (June 2006- June 2007) Crossing and cold tolerance testing (F1) of lines identified in previous step. Grow selected F2 lines in the field for F3 seed, evaluate field cold- tolerance make selections based on all traits. Combine results of Objectives 1 and 2 with prior results of this objective. Use cultured cells from derived lines to test combinations of increased metabolites on LT50. Year 5 (June 2007- June 2008) Inbred line development, cold testing, QRT-PCR analysis. Test selected lines for LT50, make further selections based on traits experience thus far shows to be useful, combine with plant lines from Objective 1 for further development. Use (cell culture) results of previous 4 years to guide plant selections for further improvement. Complete transformation studies, if warranted. 3. Milestones: A. The milestones listed below were scheduled to be completed under year one. All milestones were completed. Define genes to (micro)array, design oligos, construct arrays, preliminary hybridizations. Two small microarrays were constructed, one of 96 oligonucleotides, one with 192 oligonucleotides. Hybridizations were completed with RNA from cold stressed plants; a manuscript has been submitted. Select parents, make initial diallel crosses. Two sets of diallel crosses now have been completed; one of five parents and one of six parents. The five parent diallel cross has been evaluated for phospholipid dynamics during five weeks of cold acclimation. Establish cell culture lines from wheat cultivars known to differ in cold hardiness, become familiar with LT50 scoring on cell cultures. The impact of choline chloride and glycine betaine on the cold tolerance of cells cultured at constant room temperature has been evaluated; data are being analyzed. B. Year 2 (June 2004- June 2005) (1) Hybridization (of microarrays) with RNA from time-course exposed plants, QRT-PCR to confirm. We will complete interrogation of microarrays with RNA from winter and spring wheat plants exposed to cold temperatures. We have used QRT-PCR to assay performance of specific genes in wheat plants undergoing cold exposure and will complete this work. (2) Grow and vernalize F1s (of diallel). Do chemical analyses. Produce F2 seed in greenhouse. Much of this milestone has already been met. We have completed analysis of phospholipid dynamics during cold acclimation and will complete analysis of the data and publish the results. Predictions based on these data will be tested with the next generation (F3) of the populations. (3) Develop procedures for treating cells with specific chemicals, (concentration and length of exposure, etc.) then testing LT50. We have obtained excellent data using choline chloride and glycine betaine in this system. Over the next year, we anticipate expanding these studies to additional chemicals and integrating these trials with gene regulation studies (objective one). Year 3 (June 2005- June 2006) (1) Hybridization/QRT-PCR with RNA from a range of wheat lines; identification of lines differing in response. By year three we will have identified genes differentially responsive in wheat lines differing in cold tolerance. We will use QRT-PCR to identify specific wheat lines strongly differing in regulation of these genes. These lines will then be used to quantifying the impact of these individual genes on cold tolerance. (2) Grow F2 seed, analyze acclimation and deacclimation rates, make selections based on all traits. Seed for this study will be produced in the field in 2004-2005. Acclimation and deacclimation rates will be evaluated in controlled environment chambers. We currently are working to develop protocols for deacclimation rate studies. (3) Test range of chemicals with known metabolite induction properties, do chemical analyses, measure LT50. Work will center on the identification of specific metabolites induced, and genes concomitantly induced, in response to cold treatment and treatment with choline chloride and glycine betaine, or other suitable chemicals we identify. Year 4 (June 2006- June 2007) (1) Crossing and cold tolerance testing (F1) of lines identified in previous step. This phase of the project will concentrate on testing hypotheses and predictions developed from the previous milestones. Plants characterized for specific metabolite accumulation and cold tolerance will be crossed and the predicted cold tolerance will be tested. (2) Grow selected F2 lines in the field for F3 seed, evaluate field cold- tolerance make selections based on all traits. These F2 lines will be grown from plants with known cold tolerance and the F2 plants will have been selected to represent the range of cold tolerance and metabolite accumulation found in previous project milestones. (3) Combine results of Objectives 1 and 2 with prior results of this objective. Use cultured cells from derived lines to test combinations of increased metabolites on LT50. This objective will make use of prior information and will combine specific metabolites into single cells, and test for enhanced cold tolerance. 4. What were the most significant accomplishments this past year? A. Phospholipids play a significant role in the response of cells to stress, from both structural and signaling standpoints. Scientists in the Wheat Genetics, Quality Physiology and Disease Research Unit in Pullman, WA, evaluated the dynamics of 40 phospholipids in thirty plant populations (five parents and all possible progeny) over five weeks of cold acclimation. Evidence of heretofore unreported phospholipid signaling systems was found. This accomplishment is important because it provides a new avenue of investigation into the control of response of wheat plants to cold temperature. B. Antioxidants form a significant part of the response of plants to cold stress. Scientists in the Wheat Genetics, Quality Physiology and Disease Research Unit in Pullman, WA, measured the behavior of six members of a multigene family of a specific antioxidant (manganese superoxide dismutase) in wheat responding to cold stress. Some members of this gene family were more responsive to cold than others, indicating that specific gene forms contribute more strongly to cold tolerance than others. This finding is important because it indicates specific gene forms may be targeted for improvement of cold tolerance. C. None D. None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Identified previously unknown diversity within an antioxidant gene family in wheat and demonstrated that some members of this gene family are much more reactive to cold temperature than others. This information provides heretofore unknown targets for molecular breeding to enhance the ability of wheat plants to respond to cold stress. Identified previously unknown patterns of response of phospholipid accumulation during cold acclimation, suggestive of phospholipid signaling. This information provides the basis for investigation of novel signal cascades involved in response to cold temperature. 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? Antioxidant gene expression information has been made available to the scientific community through publication. Phospholipid production data associated with cold acclimation in wheat will be made available to the scientific community through scientific publications and presentations at professional meetings. Wheat germplasms lines developed through this project are expected to become available within five years. Transfer to the end user will be through the conventional plant breeding process, typically requiring several years to complete. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Skinner, D. Z. Lecithin in wheat and its relation to cold tolerance. Presentation to the Adams Co. (WA) Wheat Growers Association, May 18, 2004, Ritzville, WA. Skinner, D. Z. USDA-ARS & Washington State University Collaborative Wheat Research. Presentation to the Washington Association of Wheat Growers. December 10, 2003, Spokane, WA. Skinner, D. Z. USDA-ARS & Washington State University Collaborative Wheat Research. Presentation to the Dryland Agriculture Extension Agents annual meeting. December 16, 2003, Pullman, WA.

      Impacts
      (N/A)

      Publications

      • Baek, K., Skinner, D.Z. 2003. Alteration of antioxidant enzyme gene expression during cold acclimation of near isogenic wheat lines. Plant Science. 165:1221-1227.
      • Steinau, A.N., Skinner, D.Z., Steinau, M. 2003. Mechanism of extreme genetic recombinaton in weedy amaranthus hybrids. Weed Science. 51(5):696- 701.
      • Lakrod, K., Chaisrisook, C., Skinner, D.Z. 2003. Expression of pigmentation genes following electroporation of albino monascus purpureus. Journal of Industrial Microbiology. 30:369-374.
      • Lakrod, K., Chaisrisook, C., Skinner, D.Z. 2003. Transformation of monascus purpureus to hygromycin b resistance with cosmid pmocosx reduces fertility. Electronic Journal of Biotechnology. http://www.ejbiotechnology. info/content/vol6/issue2/full/3/index.html