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United States Department of Agriculture

Agricultural Research Service


Location: Plant, Soil and Nutrition Research

2011 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
Progress was made on research objectives, which fall under National Program 301. Under sub-objective 1A, ARS researchers at the Robert W. Holley Center for Agriculture & Health in Ithaca, NY, identified novel Al tolerance genes which are being exploited to increase sorghum yields on the acidic, Al toxic soils that limit agriculture on up to 50% of the world’s lands, including many developing countries. In collaboration with researchers from Embrapa Maize and Sorghum in Brazil, the first sorghum Al tolerance gene, SbMATE, was identified. SbMATE encodes a transport protein that pumps the organic acid citric acid into the soil, where the citric acid binds and detoxifies Al ions. A second protein that binds very strongly to the SbMATE protein and regulates how it functions has been found. In the absence of Al ions, this protein prevents the release of citric acid from the root. Then, when the root encounters toxic Al ions in the acid soil, this second protein binds Al ions, which causes the protein to change its structure, allowing citric acid to be transported out of the root. This regulation of SbMATE ensures that unnecessary carbon loss from the root does not occur under non-Al toxic conditions. We are taking advantage of these findings to improve productivity of sorghum grown as a major food crop in sub-Saharan Africa and investigating the possibility to increase the productivity of biofuel sorghum grown on acid soils prevalent in the southeastern U.S. Under sub-objective 1B, the first maize Al tolerance gene, ZmMATE1, was identified which is closely related to sorghum SbMATE and also is a root citric acid transporter. We have found that differential Al tolerance in maize is based on differences in expression of the ZmMATE1 gene. We have found that high ZmMATE1 expression in Al tolerant maize is due to the fact that there are 3 copies of this gene in Al tolerant lines while only 1 copy of the ZmMATE1 gene exists in Al sensitive maize. These findings may open up new avenues for improving the acid soil tolerance of maize via molecular breeding. Under sub-objective 1C, it was found that rice uses novel Al tolerance mechanisms not based on the root transport of organic acids into the soil. We are very interested in this as rice is the most important food crop in the world and very Al tolerant, yet little is known about its high Al tolerance. We have conducted “association genetics” analysis of rice Al tolerance which involves determining the level of Al tolerance in a group of 400 very diverse rice lines. We also "genotyped" (determined differences in the genetic make-up of each rice line by examining their DNA sequence using molecular markers which identify differences in DNA sequence between the rice lines) for all 400 of these lines. The association involves using a novel statistical program to identify differences in each plants DNA sequence that correlate with differences in the level of Al tolerance measured in each line. This has allowed us to identify regions of the rice genome where novel genes are involved in rice Al tolerance and we are in the process of cloning and evaluating candidate Al tolerance genes.

4. Accomplishments
1. Over 20% of the US land area and up to 50% of the world’s arable lands are highly acidic. On these acid soils, aluminum (Al) ions are dissolved into the soil solution which are toxic to and damage plant roots, thus greatly reducing crop vigor and yields. Because of the importance of this problem to agriculture worldwide, there is considerable interest in identifying genes that provide tolerance to Al toxicity in order to improve crop Al tolerance via molecular breeding and biotechnology. Rice is the most Al tolerant of all cereals and thus may be a unique genetic research for novel tolerance genes and mechanisms. However, very little is known about the genetics of rice Al tolerance. ARS researchers at the Robert W. Holley Center for Agriculture & Health at Ithaca, NY, used novel statistical genetic approaches in a large rice diversity panel to identify new regions of the rice genome involved in Al tolerance. This approach then allowed the researchers to identify novel Al tolerance genes in rice. The importance of these findings is that they open up new avenues and new candidate genes for rapidly improving the Al tolerance of rice and other cereals via molecular breeding approaches.

2. Verification that a novel protein regulates sorghum aluminum (Al) tolerance. Acid soils make up as much as 50% of the world’s soils, particularly in the tropics and subtropics where many developing countries are located. On acid soils, aluminum (Al) ions are dissolved into the soil solution and are toxic to and damage plant roots, thus greatly reducing crop vigor and yields. ARS researchers at the Robert W. Holley Center for Agriculture & Health at Ithaca, NY, had previously discovered the primary Al tolerance protein in sorghum that was shown to function by pumping the organic acid, citric acid, out of the root tip into the soil where it binds to and detoxifies the Al ion. These researchers have now discovered a second protein that binds to the citric acid pump and regulates its function. It does this by preventing citric acid to be pumped out of the root unless toxic Al ions can bind to this second protein. This ensures that unnecessary carbon loss from the root does not occur under non-Al toxic conditions. These findings are significant as they provide us with another molecular target to improve sorghum Al tolerance via molecular breeding, thus improving yields of this important staple food crop on acid soils prevalent in sub-Saharan Africa.

3. Developed novel digital imaging and computational tools to obtain 2D and 3D images of whole root systems. There is a growing realization by agricultural researchers about the importance of the architecture of plant root systems to crop traits such as nutrient and water acquisition. Researchers are discovering that the way the plant determines where in the soil profile its roots grow makes a difference; for example, in acquiring essential nutrients like phosphorous which often are in short supply to crop plants. Very little is known about the genetic regulation of root system architecture as it has been difficult to study whole root systems. Therefore, ARS researchers at the Robert W. Holley Center for Agriculture & Health at Ithaca, NY, developed a system to grow root systems of cereal roots in transparent gellan gum tubes that allow them to digitally image the root system in great detail. The researchers then use a novel software system to reconstruct the multiple images of the roots into a three dimensional model of the whole root system. This system is now being used to genetically characterize differences in root system architecture that play a role in water and nutrient uptake under limiting conditions. Subsequently, this information will be used to identify genes that control root system architecture and will provide the information plant breeders can use to carry out "root-based breeding" that should generate higher yielding cereal varieties based on superior root traits.

Review Publications
Shulz, C., Kochian, L.V., Harrison, M. 2010. Genetic variation for root architecture, nutrient uptake and mycorrhizal colonisation in Medicago truncatula accessions. Plant and Soil Journal. Available:

Famoso, A., Shaff, J., Clark, R., Mccouch, S., Kochian, L.V. 2010. Development of a novel aluminum tolerance phenotyping platform used for comparisons of cereal aluminum tolerance and investigations into rice aluminum tolerance mechanisms. Plant Physiology. 153:1678-1691.

Milner, M., Oeno, D., Naoki, Y., Yokosho, K., Zambrano, M., Kaskie, M., Ebbs, S., Jian, F., Kochian, L.V. 2011. Elevated expression of TcHMA3 plays a key role in the extreme Cd tolerance exhibited by a Cd-hyperaccumulating ecotype of Thlaspi caerulescens. Plant Journal. 66(5):852-862.

Clark, R.T., Maccurdy, R.B., Jung, J.K., Shaff, J.E., Mccouch, S.R., Aneshansley, D.J., Kochian, L.V. 2011. 3-dimensional root phenotyping with a novel imaging and software platform. Plant Physiology. 156:455-465.

Last Modified: 05/25/2017
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