Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC APPROACHES TO IMPROVING TRANSPORT AND DETOXIFICATION OF SELECTED MINERAL ELEMENTS IN CROP PLANTS

Location: Plant, Soil and Nutrition Research

2007 Annual Report


1a.Objectives (from AD-416)
1) Identify 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..
2)Describe molecular and physiological mechanisms of heavy metal/micronutrient tolerance and transport in the metal hyperaccumulator, Thlaspi caerulescens, and evaluate how these gene systems can be used for phytoremediation of metal-contaminated soils and for enhancing micronutrient nutrition of food crops.


1b.Approach (from AD-416)
1) Sorghum represents plant species where Al tolerance is a simple trait. We have recently cloned the major sorghum Al tolerance gene, AltSB, and found it is a novel solute transporter. The function of AltSB will be studied using a multifaceted approach including the effect of increased/decreased AltSB expression on the physiology of Al tolerance, association analysis correlating sequence and phenotypic variation of multiple AltSB alleles, and analysis of AltSB transporter properties when expressed in heterologous systems..
2)Maize represents a plant species where Al tolerance is a complex, quantitative trait. We have identified a number of Al tolerance QTL in maize, and will work towards cloning these QTL via a combination of gene and protein expression analysis, high resolution mapping, and analysis of candidate tolerance genes based on homology to Al tolerance genes recently cloned in sorghum and wheat..
3)An investigation of the role of hyperexpression of a suite of micronutrient and heavy metal-related genes in heavy metal hyperaccumulation in Thlaspi caerulescens will involve investigation of cis and trans factors that control micronutrient (Zn) homeostasis in the related non-accumulator, Arabidopsis thaliana, and how these elements are altered in T. caerulescens to contribute to the enhanced metal accumulation and tolerance..
4)We have recently identified several genes that play important roles in the hyperaccumulation phenotype in T. caerulescens, including a heavy metal ATPase and a protein kinase, and the functioning of these genes in heavy metal hyperaccumulation, as well as in micronutrient nutrition will be studied.


3.Progress Report
This report documents the work conducted in FY07 for 1907-21000-024-00D “Genomic approaches to improving transport and detoxification of selected mineral elements in crop plants”. For the research on sorghum aluminum (Al) tolerance genes, we previously had cloned only the second Al tolerance gene in any plant species via map-based cloning. This year we showed that this gene encodes a transporter that pumps citric acid from root cells into the soil, where the citrate complexes and detoxifies Al ions before they reach the root. We also studied gene diversity which explains the wide variation in sorghum Al tolerance. The results indicated there are multiple versions of this Al tolerance gene in different sorghum lines, but we also identified the existence of 1 or more novel sorghum Al tolerance genes. The genetic diversity analysis indicates this gene can be used in molecular breeding programs to greatly enhance sorghum Al tolerance for agriculture on acid soils.

The work in sorghum opened up new avenues for enhancing Al tolerance in other crop species. We produced transgenic wheat lines expressing the sorghum Al tolerance gene that exhibited greatly increased Al tolerance. We also identified a homolog of the sorghum tolerance gene in maize and have obtained evidence implicating this gene in maize Al tolerance.

The second objective focuses on toxic heavy metal transport in plants. A unique plant species, Thlaspi caerulescens, is the study system and can tolerate very high levels of the heavy metals zinc and cadmium in the soil, and also will accumulate these metals to very high levels in the shoots. We are working to identify the genes/mechanisms underlying this metal hyperaccumulation trait, in order to use this information to design plants better suited for the remediation of heavy metal contaminated soils. We had previously found that very high gene expression was found in this plant compared to a related non-accumulator. This hyperexpression appears to underly the hyperaccumulation triat. Thus we used a combination of gene microarray analysis and yeast complementation to identify a transcription factor that may be involved in this increased gene expression.

Research is also conducted under the following Agreements: 1907-21000-024-05S “Improving the abiotic stress tolerance, phytoremediation potential and nutritional quality of plants” (SCA with Cornell University); 1907-21000-024-04T “New approach for improving phosphorus acquisition and aluminum tolerance of plants on marginal soils” (trust fund agreement with McKnight Foundation); 1907-21000-024-07T “Improving phosphorus acquisition and aluminum tolerance of plants on marginal soils” (trust fund agreement with McKnight Foundation); and 1907-21000-024-06R “AltSB-The major aluminum tolerance gene in sorghum: Molecular/Biochemical characterization and application to improving cereal aluminum tolerance (Reimbursable agreement with USDA-NRI Competitive Grants Program). Additional details can be found in the specific reports for these agreements. The Specific Cooperative Agreement is monitored via regular meetings with the Cornell cooperators.


4.Accomplishments
A) Novel Sorghum Al Tolerance Gene Characterized: Acid soils comprise large areas of land in the US and up to 50% of the world’s potentially arable lands. On these soils, toxic forms of aluminum (Al) are solubilized from clay minerals and damage and inhibit root systems, greatly reducing crop yields. There is considerable interest in identifying genes that confer Al tolerance for use in enhancing crop acid soil tolerance, thus improving yields on acid soils. In the last year, we isolated a novel Al tolerance gene via map-based cloning in sorghum. This year, we showed that the gene encodes a citrate efflux transporter that is activated by Al, mediating release of citric acid into the soil where it binds and detoxifies Al so it does not damage the growing root tip. The significance of this discovery is that we now have a new molecular tool to improve Al tolerance not only in sorghum but other crop species, both via biotechnology and more traditional breeding approaches. This work will facilitate the achievement of components of the NP 302 Action Plan, specifically, Component I: Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement with a specific focus on Problem Statement I B: Applying Genomics to Crop Improvement, and Component 2. Biological Processes that Improve Crop Productivity and Quality, with a focus on Problem Statement 2B: Understanding Plant Interactions with Their Environment.

B) Al Tolerance Gene Shown to Have Many Forms in Sorghum: The sorghum Al tolerance gene described above in under the “Novel Sorghum Al Tolerance Gene Characterized” accomplishment is unique, in that we had previously shown that a single gene controls most of the variation in sorghum Al tolerance. This suggested there were multiple forms (alleles) of this gene in sorghum. We verified this via molecular and genetic analysis of the recently identified sorghum Al tolerance gene that showed that different sorghum lines harbor different versions of this gene. In doing so, we have identified versions of the gene that are much more effective in conferring Al tolerance and these versions will be used to enhance Al tolerance in sorghum and other cereal species (maize, wheat, barley). This work will facilitate the achievement of components of the NP 302 Action Plan, specifically, Component I: Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement with a specific focus on Problem Statement I B: Applying Genomics to Crop Improvement, and Component 2. Biological Processes that Improve Crop Productivity and Quality, with a focus on Problem Statement 2B: Understanding Plant Interactions with Their Environment.

C) Coupling of 2D Electrophoresis with liquid Chromatography and Mass Spectrometry to Resolve Multiple Proteins; We have demonstrated that the presence of multiple proteins in seemingly well resolved protein spots often confounds protein quantification based on two-dimensional electrophoresis (2DE). An alternative approach, coupling conventional 2DE with liquid chromatography and mass spectrometry (2D-GeLC-MS), has been developed that eliminates the problems introduced by the co-migration of proteins. Using this approach we have individually quantified the protein components of various gel features (spots) isolated by 2DE from protein extracts of maize root tips grown under Al free and Al treated conditions. We have found that some of the proteins detected within “up regulated” spots are actually down regulated and vice versa. The development of 2D-GeLC-MS allows for greater proteomic coverage and improved quantification which will enable us to better correlate protein expression data with the biological phenomenon under investigation. These developments will facilitate the achievement of various components of the NP 302 Action Plan, specifically, Component I: Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement with a specific focus on Problem Statement I B: Applying Genomics to Crop Improvement in that they represent a new Proteomic technology that extends genomic understanding to the level of gene products.

D) Comparison of liquid chromatography-mass spectrometry strategies; We compared the performance of two strategies for liquid chromatography-mass spectrometry analysis. The two approaches involved the orthogonal ionization methods of Electrospray (ESI) and matrix assisted laser desorption (MALDI). We observed that while these two strategies are generally complementary there are certain situations involving complex sample matrices where LC-MALDI-MS/MS has significant advantages and can increase proteomic coverage by a factor of 3. Since complex sample matrices are a commonly encountered problem in plant proteomics, analysis by LC-MALDI-MS/MS is highly recommended. This work will facilitate the achievement of various components of the NP 302 Action Plan, specifically, Component I: Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement with a specific focus on Problem Statement I B: Applying Genomics to Crop Improvement in that it represents an improved Proteomic technology that can extend genomic understanding to the level of gene products.

E) Corn Version of a Wheat Al Tolerance Gene is Not Involved in Corn Al Tolerance: The first Al tolerance gene cloned in any species was a wheat gene named ALMT1, for Al-activated malate transporter. It was shown to encode a root transporter that releases the organic acid, malate, into the soil. For our project on improving corn Al tolerance, we cloned the homolog of this wheat gene in corn. Using a combination of molecular, physiological and biophysical approaches, we showed that this corn version of the wheat tolerance gene is not involved in corn Al tolerance. Instead, it is a transporter that facilitates the movement of essential anion mineral nutrients such as nitrate and sulfate into and out of plant cells. These findings demonstrate the complexity of plant Al tolerance, showing one can not assume a tolerance gene in one species has a close homolog in another. This work will facilitate the achievement of components of the NP 302 Action Plan, specifically, Component I: Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement with a specific focus on Problem Statement I B: Applying Genomics to Crop Improvement, and Component 2. Biological Processes that Improve Crop Productivity and Quality, with a focus on Problem Statement 2B: Understanding Plant Interactions with Their Environment.


5.Significant Activities that Support Special Target Populations
The Thannhauser lab hosted a scientist from the Institute of Agricultural and Environmental Research, Tennessee State University (TSU), an 1890 University. The purpose of the visit (6/28/07-8/8/07) was to provide training in advanced proteomics techniques, access to state of the art instrumentation and software and guidance concerning the design, planning and execution of proteomics experiments. The work carried out by this scientist while at Ithaca involved the investigations of Al and salt tolerance in tomato and will be used to generate preliminary data to support a planned grant proposal to the National Research Initiative in the fall of 2007. This effort was undertaken with a view to improve both the educational and research opportunities available at an institution that has traditionally served the socially disadvantaged and historically underserved.


6.Technology Transfer

Number of patent applications filed1
Number of web sites managed1
Number of non-peer reviewed presentations and proceedings19
Number of newspaper articles and other presentations for non-science audiences2

Review Publications
Magalhaes, J., Liu, J., Guimaraes, C., Lana, U., Alves, V., Wang, Y., Schaffert, R., Hoekenga, O., Shaff, J., Klein, P., Carneiro, N., Coelho, C., Trick, H., Kochian, L.V. 2007. A member of the multidrug and toxic compound extrusion ‘MATE’ family is a major gene that confers aluminum tolerance in sorghum. Nature Genetics. 39(9):1107-1113.

Caniato, F., Guilamraes, C., Schaffert, R., Alves, V., Kochian, L.V., Borem, A., Klein, P., Magalhaes, J. 2007. Genetic Diversity for Aluminum Tolerance in Sorghum. Theoretical and Applied Genetics. 114(5):863-876.

Li, L., Lu, S., Cosman, K., Earle, E., Garvin, D.F., O'Neill, J. 2006. B-carotene accumulation induced by the cauliflower or gene is not due to an increased capacity of biosynthesis. Phytochemistry. 67:1177-1184.

Kupper, H., Seib, L.O., Kochian, L.V. A novel method for quantitative in situ hybridisation in plants reveals regulation of a zinc transporter in the cd/zn hyperaccumulator thlaspi caerulescens (ganges). Plant Journal. 50:159-175.

Papoyan, A., Pineros, M., Kochian, L.V. 2007. The effect of plant cadmium and zinc status on root and shoot heavy metal accumulation in the heavy metal hyperaccumulator, Thlaspi caerulescens. New Phytologist. 175:51-58.

Last Modified: 9/1/2014
Footer Content Back to Top of Page