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? Plants have evolved in soil environments where levels of essential and toxic metals can vary widely; hence they have developed elegant mechanisms to deal with these metals. The research detailed here uses an integrated molecular, genetic and physiological approach to identify and characterize the mechanisms underlying toxic metal tolerance and transport in plants. The work being conducted in this new ARS project that was initiated on 4/10/06 is a continuation of research that has focused on two different strategies that plants employ to deal with toxic metals, one based on metal exclusion and the other on extreme metal tolerance and hyperaccumulation. A widely studied example of the first strategy involves mechanisms plants use to tolerate toxic levels of aluminum (Al) that are found on acid soils (pH <. 5)that limit crop production on up to 50% of the world’s lands. This research involves the identification and characterization of genes and associated physiological mechanisms for aluminum tolerance in the important cereal crop species, maize and sorghum, with the long term goal of improving crop production on acid soils. Research on the second strategy involves the extreme tolerance and accumulation of heavy metals exhibited by unique plant species, termed metal hyperaccumulators. We are studying the best known of the metal hyperaccumulators, Thlaspi caerulescens; this plant species can accumulate both the heavy metal Cd and the essential micronutrient Zn to extremely high levels in the shoot. The long-term goals of the metal hyperaccumulation research are to provide the information and molecular tools that ultimately can be used to develop high biomass plants better suited for the phytoremediation of heavy metal contaminated soils. Improving the ability of farmers to cultivate crops on marginal soils such as acidic, Al toxic soils, is important as acid soils comprise over 50% of the world’s potentially arable lands, including significant land areas in the United States. While liming of acid soils ameliorates problems associated with soil acidity, this is neither an economic option for poor farmers nor an effective strategy for alleviating subsoil acidity. Marginal soils contaminated with heavy metals are also a serious worldwide problem both for human health and agriculture. Cleanup of hazardous wastes by the currently used engineering-based technologies has been estimated to cost at least $400 billion in the U.S. alone. Recently, there has been considerable interest in the use of terrestrial plants as an alternative, ‘green technology’ for the phytoremediation of surface soils contaminated with toxic heavy metals. The research to be undertaken falls under National Program 302 (Plant Molecular and Biological Processes) and addresses Component I. Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement of the NP302 Action Plan with a specific focus on Problem Statement I B: Applying Genomics to Crop Improvement. The research also includes elements of Problem Statement I A: Advancing from Model Plants to Crop Plants, and Problem Statement II B: Plant Interactions with Their Environment. 2.List by year the currently approved milestones (indicators of research progress) FY2006 – See Bridge Project 1907-21000-018-00D FY 2007 1. Complete higher resolution mapping of NIL pairs from maize IBM population to more finely delimit regions on maize genome harboring aluminum tolerance genes. 2. Conduct QTL analysis of selected additional RIL sets identified from first year analysis of parents to identify novel tolerant QTL. 3. Continue to characterize maize Al tolerance mechanisms in NIL pairs. 4. Initiate gene and protein expression profiling in maize NIL pairs from IBM population. 5. Map candidates developed from gene & protein expression analysis of maize NIL pairs and/or RIL collections. 6. Begin association analysis of candidate maize Al tolerance loci, collect SNP data and analyze results. 7. Express the sorghum Al tolerance gene, AltSB, in oocytes for electrophysiological analysis of transport properties. 8. Clone and sequence AltSB promoter region containing MITE transposon from members of sorghum diversity panel. 9. Make a series of deleted AltSB promoter-GFP constructs and test their transient expression patterns in sorghum root-tip protoplasts. Based on the results of transient gene expression patterns, cis-elements responsible for gene expression and gene regulation will be inferred. 10. Conduct Western blotting analysis to study the tissue-specific localization of the AltSB proteins. 11. Perform immunolocalization experiments to determine the subcellular localization of the AltSB protein. 12. Align and analyze promoter sequences for Zn responsive genes in Arabidopsis, to identify putative Zn responsive elements. 13. Clone and sequence promoter regions for selected genes hyper expressed in the heavy metal hyperaccumulator, Thlaspi caerulescens. 14. Align promoter sequences for selected hyper expressed genes from T. caerulescens versus Arabidopsis to search for cis elements possibly involved in hyperexpression. FY2008 1. Complete QTL analysis of maize Al tolerance in selected additional RIL sets identified from first and second year analyses of parents to identify novel tolerant QTL. 2. Continue physiological analysis of novel maize Al tolerance mechanisms in NIL pairs. Continue gene and protein expression profiling in root tips of maize NIL pairs. 3. Continue association analysis of candidate Al tolerance loci. 4. Initiate reverse genetic analysis of candidate Al tolerance loci in transgenic maize. 5. Complete development of transgenic sorghum and maize lines over expressing AltSB. 6. Begin development of AltSB knock out lines in transgenic Al tolerant sorghum. 7. Complete electrophysiological analysis of AltSB in oocytes. 8. Complete association analysis on AltSB promoter region to verify it is the Al tolerance locus. 9. Begin making chimeric AltSB promoter-GFP constructs that combine different cis-elements from Al-tolerant and Al-sensitive lines and test their transient gene expression patterns in sorghum root-tip protoplasts. 10. Conduct screens of a split ubiquitin yeast 2-hybrid library (mbSUS library) for proteins that interact with and regulate the AltSB transport protein. 11. Complete analysis of promoter sequences from the T. caerulescens and Arabidopsis to identify putative elements involved in Zn response and hyper expression. 12. Initiate in vivo transient expression analysis of promoters to identify Zn responsive elements and investigate whether differences in promoters or trans acting factors are involved in hyperexpression. 13. Initiate in vivo expression analysis of promoters in stably transformed Arabidopsis plants. 14. Clone E2F homologs from Arabidopsis and Thlaspi arvense and express in yeast ZHY6 mutant to determine if they function differently than Thlaspi caerulescens E2Fs. 15. Over express T. caerulescens E2Fs in Arabidopsis to see if any aspect of the hyper accumulation phenotype is re-constituted. FY2009 1. Continue physiological analysis of novel maize Al tolerance mechanisms in NIL pairs. 2. Continue gene and protein expression profiling in root tips of maize NIL pairs. 3. Continue association analysis of candidate Al tolerance loci. 4. Continue reverse genetic analysis of candidate Al tolerance loci in transgenic maize. 5. Begin detailed molecular and physiological analysis of maize Al tolerance genes identified from association and reverse genetics analysis. 6. Continue physiological analysis of transgenic sorghum and maize lines over expressing AltSB. 7. Continue development of AltSB knock out lines in transgenic Al tolerant sorghum. 8. Utilize association tools to identify novel, elite alleles of AltSB. 9. Make selective AltSB promoter-GFP constructs for stable transformation of sorghum plants. 10. Continue analysis of proteins interacting with AltSB, including in vitro pull-down assays to verify proteins that interact with and possibly regulate the AltSB protein. 11. Map the genes encoding proteins that interact with AltSB to mapping populations where tolerance is not linked to AltSB to see if they map to regions of the genome harboring novel Al tolerance loci. 12. Continue in vivo transient expression analysis of T. caerulescens and Arabidopsis promoters for Zn-responsive genes with a focus on selected promoter regions identified from initial experiments. 13. Continue in vivo expression analysis of promoters in stably transformed Arabidopsis plants. 14. Initiate in vivo expression analysis of promoters in stably transformed Thlaspi caerulescens plants. 15. Initiate yeast 1-hybrid screen for T. caerulescens and Arabidopsis transcription factors that interact with ZNT1/ZIP4 and HMA4 promoters as bait. 16. Conduct similar yeast 1 hybrid experiments with tandem copies of promoter elements identified from previous promoter analyses as bait. FY2010 1. Complete physiological analysis of novel maize Al tolerance mechanisms in NIL pairs. 2. Complete gene and protein expression profiling in root tips of maize NIL pairs. 3. Continue association analysis of candidate Al tolerance loci. 4. Continue reverse genetic analysis of candidate Al tolerance loci in transgenic maize. 5. Continue detailed molecular and physiological analysis of maize Al tolerance genes identified from association and reverse genetics analysis. 6. Continue physiological analysis of transgenic sorghum and maize lines over expressing AltSB. 7. Begin physiological analysis of AltSB knock out lines in transgenic Al tolerant sorghum. 8. Continue to utilize association tools to identify novel, elite alleles of AltSB. 9. Begin field testing of transgenic sorghum and maize lines overexpressing AltSB. 10. Develop markers for molecular breeding of elite AltSB alleles to improve sorghum Al tolerance. 11. Continue to generate and analyze sorghum transgenic plants for promoter activity. 12. Select homozygous transgenic plants and analyze their gene expression patterns for the reporter gene (GPF expression patterns) to identify promoter regions critical for AltSB expression. 13. Analyze and characterize candidates for novel sorghum Al tolerance genes identified in previous year’s research. 14. Complete analysis of proteins interacting with AltSB, including in vitro pull-down assays to verify proteins that interact with and possibly regulate the AltSB protein. 15. Continue to map the genes encoding proteins that interact with AltSB to mapping populations where tolerance is not linked to AltSB to see if they map to regions of the genome harboring novel Al tolerance loci. 16. Complete in vivo expression analysis of Thlaspi caerulescens and Arabidopsis promoters in stably transformed Arabidopsis and Thlaspi plants. 17. Continue analysis of candidate T. caerulescens transcription factors begun in previous year via electromobility shift assays, DNase 1 footprinting assay, and modified yeast 1-hybrid using promoter sequences and transcription factor as bait and prey. 4a.List the single most significant research accomplishment during FY 2006. Because research for this new CRIS project was only conducted for approx 5 months in FY2006, the information on major and additional accomplishments for FY2006 milestones for questions 4A and B are contained in the annual report for the bridge CRIS project that terminated on 4/10/06, CRIS project 1907-21000-018-00D. 4b.List other significant research accomplishment(s), if any. None. 4c.List significant activities that support special target populations. None. 4d.Progress report. This report serves to document research conducted under a trust fund agreement between ARS and USDA-NRI entitled “Molecular regulation of heavy metal and micronutrient homeostasis and hyperaccumulation” 1907-21000-024-03T. During the current year, we investigated the heavy metal (Zn/Cd) hyperaccumulator, Thlaspi caerulescens, to elucidate the mechanisms underlying the extreme heavy metal tolerance and hyperaccumulation of micronutrients and heavy metals expressed by this plant species. It is not known whether the metal tolerance and hyperaccumulation traits are expressed partly at the cellular level, or if they require the functioning of many different cell types and tissues in the plant. To test this, single suspension cell cultures of Thlaspi and the related non-accumulator, Arabidopsis thaliana, were developed and characterized for heavy metal tolerance and accumulation. From these studies it was found that single Thlaspi cells are more metal tolerant, and exhibit differences in metal transport. Surprisingly, the Thlaspi cells actually accumulated less Cd and Zn than did Arabidopsis suspension cells. It is hypothesized that since Thlaspi very efficiently moves metals from the root to the final site of metal storage, leaf epidermal cells, the suspension cells represent cells of the transport pathway, where it is advantageous to keep the metals in a more mobile pool in the plant apoplast. This project terminated on 8/31/2006. This report serves to document research conducted under a trust fund agreement between ARS and McKnight Foundation competitive grant entitled “New approach for improving phosphorus acquisition and aluminum tolerance of plants on marginal soils” 1907-21000-024-04T. During the current year, we verified with near isogenic lines, that the 5 Al tolerance QTLs we identified the previous year in maize were bona fide Al tolerance QTLs. We also identified a number of candidate maize Al tolerance genes, including several that may underlie a novel tolerance mechanism associated with changes in root cell wall composition. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the Department of Plant Biology at Cornell University entitled “Improving the abiotic stress tolerance, phytoremediation potential and nutritional quality of plants” 1907-21000-024-05S. With regards to research on sorghum and maize aluminum tolerance, in this year we characterized and verified that the candidate aluminum (Al) tolerance gene in sorghum, AltSB, was in deed a major Al tolerance gene. AltSB encodes an Al-activated citrate efflux transporter that is the basis for the major physiological Al tolerance mechanism in sorghum. The research on heavy metal hyperaccumulation in Thlaspi caerulescens continues to focus on the molecular basis for the extreme heavy metal/micronutrient accumulation in the shoots of Thlaspi caerulescens. We have identified cis (promoter regions) and trans factors (transcription factors) that may help drive the hyperexpression of a number of metal-related genes and appears to underlie metal hyperaccumulation, and in this year we identified several candidate cis and trans-acting factors that will be characterized in the next several years to elucidate the roles these factors play in hyperexpression. 5.Describe the major accomplishments to date and their predicted or actual impact. This new CRIS project was initiated on 4/10/06, and the research accomplishments for this research are listed in the answers to Question 4B of the bridge CRIS annual report, CRIS project 1907-21000-018-00D. 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? This new CRIS project was initiated on 4/10/06. Please see the answers to question 6 for bridge CRIS annual report, CRIS project 1907-21000-018-00D. 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). This new CRIS project was initiated on 4/10/06. Please see the answers to question 7 for bridge CRIS annual report, CRIS project 1907-21000-018-00D.