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United States Department of Agriculture

Agricultural Research Service

Research Project: CHARACTERIZATION AND ENHANCEMENT OF PLANT RESISTANCE TO WATER-DEFICIT AND THERMAL STRESSES

Location: Plant Stress and Germplasm Development Research

2006 Annual 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?
Environmental stresses on plants consistently reduce crop production in all agricultural areas. Deficits of available soil water caused by untimely rainfall and temperature extremes are annual occurrences in the rain fed agricultural areas of the United States. Soil water deficits and temperature extremes are two stresses that cause major impact on growth and yield.

We are identifying metabolic limitations responsible for reduced plant performance under water and temperature stresses. We are identifying and characterizing natural defense mechanisms used by plants to reduce water and temperature stress inhibition of growth and development. We are assessing the impact of temperature and water stresses on germplasm collections, developing improved management systems for existing germplasm, and developing transformation technology for germplasm improvement.

Comparison of average crop yields, with reported record yields, has shown that the major crops grown in the U.S. exhibit annual average yields three to seven-fold lower than record yields because of unfavorable environments. Analysis of yields from corn, wheat, soybeans, sorghum, oats, barley, potatoes, and sugar beets revealed that the average yield represented only 22% of the mean record yield. Crops with economically valuable reproductive structures showed the greatest discrepancy between average and record yields. Those crops having marketable vegetative structures exhibited approximately three-fold reductions in yield. These data suggest that plants have high productivity potentials, but are operating well below their genetic potential. Evaluation of crop losses between 1948 and 1989 by the Federal Crop Insurance Corporation showed that on average, 69% of insurance indemnities could be attributed to drought and excess heat in barley, canning beans, corn, forages, oat, peanut, rye, safflower, soybean, and wheat. Improved management and germplasm are essential if U.S. farmers are to remain competitive in the world market place.

This project is assigned to the Plant Biological and Molecular Processes National Program 302. The program component entitled Biological Processes that Determine Plant Productivity and Quality states that crop production potential is much greater than currently realized, in part because crop plants do not use, or they inefficiently use, all of the available resources. Specifically, our research addresses Problem Area IIb Plant Tolerance to Environmental Stresses of the NP302 action plan. Our research concentrates on long-term discovery research to improve the manipulation of genes and gene expression for agricultural purposes. Our research goals include, but are not limited to, identifying sources of new genes; recognizing and modifying important genes and the mechanisms for regulation of their expression; characterizing and manipulating the cell signals that govern the patterns of gene activities; altering the processes of recombination to improve transgene expression and provide specificity to its genomic location; and tailoring the temporal, spatial, and environmental regulation of transgenes to meet specific needs. Under this National Program our research is aimed at developing an understanding of the continuum from DNA to phenotype and the mechanisms that provide that continuum.


2.List by year the currently approved milestones (indicators of research progress)
Year 1 (FY2006) Generation of cDNA libraries from peanut tissue exposed to abiotic stress. Fabrication of anonymous peanut cDNA arrays for expression studies. Physiological analysis of water-deficit stress on field-grown peanut.

T-DNA insertion lines will be analyzed using the chlorophyll accumulation biossay. Phenotypes of identified mutants will be confirmed and attempts will be made to obtain homozygous populations.

EMS-induced mutations in ESK1 gene will be isolated through the Arabidopsis Tilling Project.

Cotton germplasm will be screened for water deficit and thermal stress using chlorophyll fluorescence assay. Variability in growth of root systems will be analyzed.

Extraction and assay procedure development. Field water deficit study with initial suite of enzymes.

Development of standard operations procedures in extraction, detection and analysis of different plant metabolites.

1,000 F2 seeds from a cross between a heat tolerant and a heat sensitive Arabidopsis ecotype will be grown in a manner assuring identify preservation. Single seed descents will be used for subsequent generations.

Transcript profiling studies in non-fiber cotton tissues will be initiated. Expression studies in drought and temperature stressed tissues.

Existing constitutive synthetic promoters will be further tested; stable transgenics generated and representative promoters chosen for further analysis.

A collection of virally encoded suppressors of gene silencing will be modified for expression in plants. Functional testing will begin.

Five expression cassettes will be designed for analysis of candidate genes identified in expression studies. Constructs will be used for cotton transformation via Agrobacteria.

Build promoter-reporter constructs and test function transiently.

Generate transgenic tobacco plants over-expressing the PAP2. Self transgenics for homozygotic, highly pigmented plants.

Year 2 (FY 2007)

Expression profiling studies in stressed tissues will be performed to identify stress responsive cDNAs. Confirmation of differential expression and quantitative expression measurements of selected known genes will be evaluated by real-time PCR.

Additional T-DNA insertion lines will be analyzed using the chlorophyll accumulation bioassay. Homozygous mutant lines will be analyzed for temperature sensitivity using alternative thermotolerance assays.

All the recovered mutants will be sequenced to confirm the mutation.

Additional cotton germplasm will be screened for water deficit. Analysis of variability in root growth will continue.

Field water deficit study of an initial suite of enzymes will begin.

Complete initial analysis of targeted plant metabolites using selected germplasm and identify candidate metabolites that are tightly associated with drought tolerance and temperature stresses.

1,000 F4 seeds from a cross between a heat tolerant and a heat sensitive Arabidopsis ecotype will be grown in a manner assuring identify preservation. Single seed descents will be used for subsequent generations.

Transcript profiling in drought-stressed peanut will use systems approaches to identify stress response genes in diverse species.

Analysis of constitutive promoters will continue and synthetic inducible promoters produced using previously identified elements will be initiated.

Suppressors will be used to study various aspects of post-transcriptional gene silencing (PTGS) in different plant species.

Additional expression cassettes will be designed, followed by cotton transformation. Analysis of transgenic plants created during year one, inlcuding response to drought and high temperature.

Generate stable transgenics with selected promoter-reporter constructs.

Test the impact of hyper-pigmentation on plant growth. Test alternative regulatory genes and gene combinations for effective anthocyanin induction.

Year 3 (FY 2008)

cDNA identification and sequencing of candidate cDNAs from expression studies. Biochemical and metabolomic analysis of peanuts from field studies and greenhouse experiments.

Additional T-DNA insertion lines will be analyzed using the chlorophyll accumulation bioassay. Homozygous mutant lines will be analyzed for temperature sensitivity using alternative thermotolerance assays.

Alteration in freezing tolerance and growth phenotype of all the recovered mutants of ESK1 will be analyzed.

Selected germplasm will be screened for water deficit and thermal stress under field conditions. Studies will be conducted to evaluate germplasm for recovery from water deficit and thermal stress.

Field water deficit study with enzymes and metabolites identified from cotton gene and metabolite profiles will continue.

Verify previous results and complete comparative analysis of plant metabolites. Start testing application of metabolite screens on target germplasm and/or breeding material.

1,000 F6 seeds from a cross between a heat tolerant and a heat sensitive Arabidopsis ecotype will be grown in a manner assuring identify preservation. Single seed descents will be used for subsequent generations.

Confirmation of stress responsive expression patterns and correlation of expression data with protein and metabolite data. Association of expression and phenotype.

Analysis of constitutive promoters will conclude; synthetic inducible promoters will be tested transiently; inducible elements from stress responsive promoters will be isolated based upon mutant and microarray analysis.

PTGS analysis will continue.

Analysis of transgenic plants for abiotic stress response will continue via gene expression studies on transgenic plants.

Select homozygous transgenic plants and study gene expression patterns and regulation in response to abiotic stresses using full-length promoters.

Selected regulatory genes and/or gene combinations introduced into stable transgenics and pigmentation examined.

Year 4 (FY 2009)

Generation of full-length candidate genes identified in expression and sequencing studies. Bioinformatic merger of expression data from the public domain and in-house expression studies to create a database of stress-responsive genes.

Homozygous mutant lines will be analyzed for temperature sensitivity using alternative thermotolerance assays.

Mutants with a significant increase in freezing tolerance but little or no growth retardation will be identified and analyzed.

Selected germplasm will be screened for water deficit and thermal stress under field conditions. Studies will be conducted to evaluate germplasm for recovery from water deficit and thermal stress.

Field water deficit study of enzymes and metabolites identified from cotton gene and metabolite profiles will continue.

Screen potential stress tolerant germplasm and/or breeding lines using selected metabolic profiling approach. Verified results using other biological stress assays.

F8 lines from a cross between a heat tolerant and a heat sensitive Arabidopsis ecotype will be analyzed for heat tolerance using the chlorophyll accumulation bioassay.

Confirmation of stress responsive expression patterns will be performed and correlation of expression data with protein and metabolite data will be evaluated.

Analysis of synthetic inducible promoters will continue as stable transgenics; promoters with new elements will be tested transiently.

Different classes of suppressors will be used to examine how environmental factors (heat, drought, light) impact PTGS.

Yield analysis and fiber quality study of selected transgenic lines for field studies will be performed.

A promoter deletion series will be constructed, tested transiently, and corresponding transgenic plants will be generated.

Stress-inducible promoter-regulator combinations will be built and tested for induction and stability of pigmentation.

Year 5 (FY 2010)

Analysis of transgenic and/or mutant germplasm with altered expression of selected candidate genes.

Homozygous mutant lines will be analyzed for temperature sensitivity using alternative thermotolerance assays.

Optimized mutation pattern that enhances freezing tolerance, but has little effect on growth will be identified.

Information on enzyme responses to field and non-field water deficits will be collated.

Incorporate targeted metabolic profiling approach into research projects using biological, genomic, proteomic and/or other approaches. Development and characterization of stress tolerant plant germplasm.

QTLs for heat tolerance will be identified.

Bioinformatic assembly of key stress response pathways and mechanisms will be performed.

Analysis of synthetic inducible promoters will continue on selected promoters.

Stress effects on silencing will be examined in stable transgenics.

Yield analysis and fiber quality study of selected transgenic lines.

Characterize transgenic plants in order to identify short promoter regions that confer high temperature specific gene expression.

Selected stress-inducible promoter-pigmentation regulator combinations will be tested in stable transgenics.


4a.List the single most significant research accomplishment during FY 2006.
Heat Tolerance Genes Identified: Scientists within the Plant Stress and Germplasm Development Unit in Lubbock, Texas, have identified two genes (digalactysyldiacylglycerol synthase and AtFtsH11 protease) contributing to basal heat tolerance in plants. This discovery advances our understanding on the many cellular components impacting heat tolerance. The scientists evaluated EMS-derived and tDNA mutants of Arabidopsis for high temperature sensitivity. This discovery, in combination with additional gene discoveries, will allow scientists to develop more heat-tolerant crops. (NP302, Performance Measure 1.2.7, Subobjective 1a).


4b.List other significant research accomplishment(s), if any.
Gene Silencing in Plants Identified: Scientists within the Plant Stress and Germplasm Development Unit in Lubbock, Texas, have developed an in vivo assay system for analyzing transient gene expression in tobacco leaves infused with Agrobacterium tumefaciens. This technique provided a needed tool for evaluating post-transcriptional gene silencing (PTGS). The scientists used the in vivo transient assay to examine the effect on PTGS of factors such as: promoter strength; incubation temperature and double-stranded RNA production. Results from these assays provide insight into the mechanism(s) used by plants to trigger and maintain PTGS. (NP302, Performance Measure 1.2.6, Subobjective 3a).


4c.List significant activities that support special target populations.
None


5.Describe the major accomplishments to date and their predicted or actual impact.
All accomplishments made under this project are fully consistent with relevant milestones listed in the Project Plan, and with the relevant research components as defined in the National Program 302 Action Plans. Accomplishments under this project contribute to the achievement of ARS Strategic Plan Goal 1, Objective 2, Performance Measures 6 and 7, in that project accomplishments contribute substantially to attainment of the Agency FY 2007 target of information will be available for more species to guide manipulation of regulatory metabolic processes that influence plant growth, product composition, product quality, and profitability; and identified important quantitative trait loci that govern key agronomic traits for a variety of crop species and made progress on sequencing gene-rich regions of a limited number of plant genomes.

Heat Tolerance Genes Identified: Scientists within the Plant Stress and Germplasm Development Unit in Lubbock, Texas, have identified two genes (digalactysyldiacylglycerol synthase and AtFtsH11 protease) contributing to basal heat tolerance in plants. This discovery advances our understanding on the many cellular components impacting heat tolerance. The scientists evaluated EMS-derived and tDNA mutants of Arabidopsis for high temperature sensitivity. This discovery, in combination with additional gene discoveries, will allow scientists to develop more heat-tolerant crops.

Gene Silencing in Plants Identified: Scientists within the Plant Stress and Germplasm Development Unit in Lubbock, Texas, have developed an in vivo assay system for analyzing transient gene expression in tobacco leaves infused with Agrobacterium tumefaciens. This technique provided a needed tool for evaluating post-transcriptional gene silencing (PTGS). The scientists used the in vivo transient assay to examine the effect on PTGS of factors such as: promoter strength; incubation temperature and double-stranded RNA production. Results from these assays provide insight into the mechanism(s) used by plants to trigger and maintain PTGS.


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?
Arabidopsis mutants containing defective heat tolerance genes have been provided to the Arabidopsis stock center for distribution to other scientists.


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).
None


Review Publications
Cazzonelli, C.I., Velten, J.P. 2006. An in vivo luciferase-based transient assay system using agrobacteria-infiltration: Implications for post-transcriptional gene silencing. Planta. 224(3):582-597.

Light, G.G., Roxas, V.P., Mahan, J.R., Allen, R.D. 2005. Transgenic cotton (Gossypium hirsutum L.) seedlings that express a tobacco glutathione S-transferase fail to provide improved stress tolerance. Planta. 222(2):346-354.

Mahan, J.R., Burke, J.J., Wanjura, D.F. 2005. Determination of temperature and time thresholds for BIOTIC irrigation of peanuts on the Texas Southern High Plains. Irrigation Science. 23:145-152.

Mahan, J.R., Mauget, S.A. 2005. Antioxidant metabolism in cotton seedlings exposed to temperature stress in the field. Crop Science. 45:2337-2345.

Burke, J.J., Chen, J. 2006. Cellular and molecular basis of plant tolerance to heat stress. In: Huang, B, editor. Plant-Environment Interactions. 3rd Edition. Boca Raton, FL: CRC Press. p. 27-46.

Mahan, J.R., Light, G., Dawson, K., Dotray, P. 2006. Thermal dependence of bioengineered glufosinate tolerance in cotton. Weed Science. 54:1-5.

Chen, J., Burke, J.J., Xin, Z., Xu, C., Velten, J.P. 2006. Characterization of the arabidopsis thermosensitive mutant ATTS02 reveals an important role for galactolipids in thermotolerance. Plant Cell and Environment. 29(7):437-1448.

Chen, J., Burke, J.J., Velten, J.P., Xin, Z. 2006. FtsH11 protease plays a critical role in Arabidopsis thermotolerance. The Plant Journal. 48(1):73-84.

Kresovich, S., Barbazuk, B., Bedell, J., Borrell, A., Buell, R., Burke, J.J., Clifton, S., Cordonnier-Pratt, M., Cox, S., Dahlberg, J., Erpelding, J.E., Fulton, T.M., Fulton, B., Fulton, L., Gingle, A., Goff, S., Hash, C., Huang, Y., Jordan, D., Klein, P., Klein, R.R., Magalhaes, J., McCombie, R., Moore, P.H., Mullet, J.E., Ozias-Akins, P., Paterson, A.H., Porter, K., Pratt, L., Roe, B., Rooney, W., Schnable, P., Steely, D.M., Tuinstra, M., Ware, D., Warek, U. 2005. Toward sequencing the sorghum genome: A US National Science Foundation-sponsored workshop report. Plant Physiology. 138(4):1898-1902.

Last Modified: 7/23/2014
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