Progress 10/01/10 to 06/27/13
Outputs
Progress Report 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. 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. As the project terminated this year, the Progress Report summarizes the life of the project. For research on maize aluminum (Al) tolerance, ARS researchers in Ithaca, NY, identified and characterized the first maize Al tolerance gene. This gene, ZmMATE1, encodes a root citrate transporter that is activated by toxic Al ions in highly acidic soils and releases citrate from the root to the soil, where it binds and detoxifies Al ions. In collaboration with scientists from Embrapa in Brazil, genetic markers for this gene were used to improve maize production on acid soils via molecular plant breeding. It was discovered that very Al tolerant maize lines have three functional copies of the ZmMATE1 gene while less Al tolerant maize only have 1 copy. This is one of the first examples where variation in gene copy number controls an agronomically important trait and this information will assist breeders in generating more Al tolerant maize lines. For the research on sorghum Al tolerance, ARS researchers in Ithaca, NY, cloned one of the first plant Al tolerance genes, SbMATE. It was shown that SbMATE encodes a root citrate transporter that functions similarly to ZmMATE1 described above. SbMATE is regulated by Al ions both in increasing gene expression and activating the citrate transport protein. We have shown that activation of gene expression involves specific DNA sequences within the SbMATE gene as well as a second protein that actually activates high SbMATE gene expression. We then identified a 3rd protein that binds to the SbMATE protein and regulates the Al activation of citrate release from the root. Thus we have generated a molecular toolbox of 3 genes which are enabling us, in collaboration with Embrapa in Brazil and sorghum breeders in Africa, to improve sorghum production on the acid soils that limit crop production in many areas of sub-Saharan Africa. For the research on genetic mapping of root system architecture (RSA), which is important for acquisition of limiting nutrients like N, P and water, novel techniques were developed that enable the imaging of plant roots and the generation of 3D reconstructions of the entire root system. Novel software then automatically computes a number of different RSA traits that could play a role in more efficient nutrient acquisition. The system was used to genetically map rice RSA traits which is leading the way to the cloning of several genes underlying potentially important RSA traits. This system was recently improved by changing from growing the roots of plants in gel cylinders (which hold the 3D structure of the root systems) to a system where roots are grown in nutrient solution using a series of fine plastic grids spaced at vertical intervals, allowing the roots to grow freely while the grid maintains the 3D architecture of the root systems. This new growth system opens up many more avenues for our RSA research, as it enables the study of a much larger number of crop species and the imaging of root systems of significantly older plants which allows for the study of more fully developed root systems. Significant Activities that Support Special Target Populations: ARS researchers at Ithaca, New York have hosted three graduate students and their professor from the Department of Agricultural Sciences at Tennessee State University, an 1890 College with a tradition of educating under representative minorities in the sciences. The visitors were supported by an AFRI Strengthening Grant and worked with Ithaca ARS scientists from May 23-July 28, 2013. They learned useful skills in protein chemistry and proteomics including multiplexed stable isotope labeling techniques, the operation high performance separations systems, the operation of state of the art mass spectrometers and the use of high throughput data analysis software. They were also able to attend a two day, �hands-on� workshop on proteomics presented at Cornell University in which ARS scientists participated as both lecturers and subject matter experts. Aside from their programed exposure to state-of-the-art proteomics they were able to meet with other location scientists and learn about state of the art with regards to mineral analysis of plant, soil and animal tissue via inductively-coupled plasma mass spectrometry and novel root architecture imaging technologies. These experiences enhanced their technical background, scientific knowledge, and problem solving skills. Furthermore the research carried out will be incorporated in their graduate degree theses. Manuscripts are being prepared concerning the research carried out this year and the two manuscripts that were highlighted in last year�s report were submitted to a peer reviewed journal and have been accepted for publication. ARS researchers at Ithaca, New York hosted two summer undergraduate interns from the University of Puerto Rico and Green Mountain University, Vermont. Both are minority (Hispanic) students and they participated as part of a collaborative summer program between the USDA, Boyce Thompson Institute (BTI), and Cornell University funded by the National Science Foundation to involve under-represented minorities in plant science research. The students worked on a project on cereal aluminum tolerance over a 10 week period and then they each presented a talk on their research at the 12th annual BTI-Cornell-USDA Plant Genome Research Symposium held at the end of their research tenure. One of the students was awarded a prize for his presentation from a panel of Cornell, BTI and ARS judges. During this period of time the students learned useful skills in molecular biology and functional characterization of proteins using heterologous expression systems, which enhanced their technical background, scientific knowledge, and problem solving skills. Accomplishments 01 Identification of the mechanisms regulating the function of plant aluminum (Al) tolerance proteins. For many human, animal and plant membrane transport proteins (proteins embedded in cellular membranes that mediate the movement of ions and solutes across those membranes), their transport activity is regulated by secondary modification of the protein. One type of such modification is a process by which a phosphate group is added to specific amino acids in the protein (a process known as phosphorylation) thereby turning the protein�s transport activity on and off. ARS researchers at Ithaca, New York have identified the specific proteins which phosphorylate the citrate transporter essential for providing to corn and sorghum plants tolerance to the toxic levels of Al found on acid soils that comprise up to 20% of the soils in the US and as much as 40% of the world�s potentially arable lands. This discovery will enable researchers to gain a better understanding of the regulatory networks involved in switching on or off the transporters underlying Al tolerance. This, in turn, will provide researchers with new research avenues to modify these transporters via protein engineering in order to enhance crop Al tolerance and increase cereal crop yields on acid soils in the US and also on the acid soils that limit crop production in the tropics and sub-tropics where many developing countries are located. 02 Improved yields from sorghum grown on high acid soils. Acid soils make up as much as 40% of the world�s soils, particularly in the tropics and subtropics where many developing countries are located. Also significant areas in the eastern and southern US have highly acidic soils. On these acid soils a form of aluminum (Al), the Al3+ ion, is solubilized from clay minerals and is highly toxic to plant roots. Thus many crops have reduced yields on these acid soils because they sustain significant damage to their root systems inhibiting their ability to take up water and nutrients. ARS researchers at Ithaca, New York, have built upon their previous discovery of a gene in sorghum encoding a protein that transports citric acid from the growing roots into the acid soil where the citric acid binds and detoxifies Al3+ ions, allowing the sorghum roots to grow in this toxic environment. They now have identified and verified the functioning of a novel mechanism of regulation of the sorghum Al tolerance protein, SbMATE. A second protein, SbMBP (SbMATE binding protein) has been identified that binds very strongly to the SbMATE protein and regulates its function. We found that the binding of SbMBP to SbMATE blocks citrate transport and that SbMBP is an Al sensor and when it binds Al ions, it is released from the SbMATE protein, allowing the transport of citrate out of the root. This regulation of SbMATE ensures that unnecessary carbon loss from the root does not occur under non-Al toxic conditions, as citrate release from the root is a significant cost to the plant. In collaboration with Embrapa Maize and Sorghum, Brazil, we are using 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. 03 New protocols developed for isolating proteins from plant tissues. ARS researchers at Ithaca, New York have developed new protocols for extracting proteins from plant tissues in a nearly quantitative manner. Understanding how the changes in protein concentration impact the metabolic pathways and physiology that comprise the biology of plants is key to our understanding of a plant�s response to environmental stress as well as to the expression of its agronomic traits. Many studies of global protein expression designed to reveal the details of these complex biological processes begin with protein extracts that contain less than 20% of the subject tissue�s protein content, leaving one to speculate concerning the biological relevance of the sample. By applying a series of complementary extraction protocols it was shown that it is possible to achieve nearly quantitative extraction of protein from a variety of plant tissues, thus, ensuring the biological relevance of the sample. 04 Improved methods for studying glycoproteins in plants. ARS researchers at Ithaca, New York, have developed improved methods for analyzing sugar-containing proteins (called glycoproteins) by incorporating several pre-fractionation steps in order to reduce the complexity of the sample mixture to be analyzed. The method takes advantage of our knowledge of the specific binding of a family of proteins known as lectins for sugar containing proteins. The specificity of various lectins towards sugar-containing proteins varies widely from the broad specificity type (where the lectin will bind any sugar containing protein) to the narrow specificity type where the lectin only binds to proteins containing a specific type of sugar molecule. By varying the nature of the lectin used one can separate a complex mixture of glycoproteins into a small number of less complex fractions. By analyzing these less complex mixtures individually it is possible to identify a larger number of glycoproteins than could be found in the original mixture. The addition of sugars to proteins is a very common modification of plant proteins that is known to alter their function. Thus, the new fractionation method will improve our ability to detect and identify sugar containing proteins and lead to a better understanding of the role of this protein modification in determining important plant traits and possibly can be used to improve specific traits in plant species. 05 Improved system for imaging whole root systems and studying root system architecture. ARS researchers at Ithaca, New York had previously developed a novel platform, RootReader3D, for high throughput imaging of root systems in 2D and reconstructing those images into a 3D representation of the root system. The RootReader 3D system then automatically quantifies 20 different root system architecture (RSA) traits that quantify both total root system growth and growth of individual root types, as well as the shape and form of the entire root system. This allows researchers to quantify RSA traits associated with deeper root systems or more shallow and wider root systems that might be more effective for acquiring nutrients such as water and nitrogen that move quickly through the soil (deeper root systems) or nutrients such as phosphorous that interact more with soil components and thus tend to move slowly through the soil and accumulate in the top soil (shallow root systems). Our initial system used plants with their roots grown in glass cylinders containing gelled nutrient media, in order to capture the 3D RSA. We found that roots of certain plant species did not grow optimally in this gel media and the research was limited to fairly small volume cylinders and thus young root systems. Therefore an improved growth system was developed where roots are grown in nutrient solution using a series of fine plastic grids spaced at vertical intervals, allowing the roots to grow freely while the grid maintains the 3D architecture of the root systems. This new growth system opens up many more avenues for our RSA research, as it enables the study of a much larger number of crop species, the imaging of root systems of significantly older plants which allows for the study of more fully developed root systems.
Impacts
(N/A)
Publications
- Milner, M., Seamon, J., Kochian, L.V. 2013. Transport properties for members of the ZIP family in plants and their role in Zn and Mn homeostasis. Journal of Experimental Botany. 64(1):369-381.
- Jung, H., Gayomba, S.R., Rutzke, M., Kochian, L.V., Vatamaniuk, O.K. 2012. COPT6 is a plasma membrane transporter that functions in copper homeostasis in Arabidopsis and is a novel target of SQUAMOSA promoter binding protein-like 7. Journal of Biological Chemistry. 287:33252-33257.
- Liang, C., Pineros, M., Tian, J., Yao, Z., Sun, L., Liu, J., Shaff, J., Liao, H., Kochian, L.V. 2013. Low pH, aluminum and phosphorus coordinately regulate malate exudation through GmALMT1 to improve soybean adaptation to acid soils. Plant Physiology. 161:1347-1361.
- Liu, J., Sivaguru, M., Kochian, L.V. 2013. Targeted expression of SbMATE in the root distal transition zone is responsible for sorghum aluminum resistance. Plant Journal. DOI: 10.1111/tpj.12290.
- Wang, Y., Yang, Y., Fei, Z., Yuan, H., Fish, T., Thannhauser, T.W., Mazourek, M., Kochian, L.V., Wang, X., Li, L. 2013. Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development. Journal of Experimental Botany. 64(4):949-961.
- Maron, L., Guimareas, C., Matias, K., Albert, P.S., Birchler, J.A., Bradbury, P., Buckler IV, E.S., Coluccio, A.E., Danilova, T.V., Kudrna, D., Magalhaes, J.V., Pineros, M., Schatz, M.C., Wing, R., Kochian, L.V. 2013. Aluminum tolerance is associated with higher MATE1 gene copy-number in maize. Proceedings of the National Academy of Sciences. 110(13):5241-5246.
- Melo, J., Lana, U., Pineros, M., Alves, V., Guimaraes, C., Liu, J., Zheng, Y., Zhong, S., Fei, Z., Maron, L., Schaeffert, R., Kochian, L.V., Magalhaes, J. 2013. Incomplete transfer of accessory loci influencing SbMATE expression underlies genetic background effects for aluminum tolerance in sorghum. Plant Journal. 73(2):276-288.
- Clark, R., Famoso, A., Zhao, K., Shaff, J., Bustamante, C., Mccouch, S., Aneshansley, D., Kochian, L.V. 2013. High-throughput 2D root system phenotyping platform facilitates genetic analysis of root growth and development. Plant Cell and Environment. 36(2):454-466.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. Under sub-objective 1A, which deals with maize aluminum (Al) tolerance, ARS researchers at the Robert W. Holley Center for Agriculture & Health in Ithaca, NY, studied the regulation of ZmMATE1 expression, the first identified maize Al tolerance gene which is closely related to our sorghum Al tolerance gene, SbMATE. Like SbMATE, it is a root transporter which mediates citric acid release from the root tip into the soil where the citric acid binds and detoxifies Al ions in the soil, allowing the root to grow. It was discovered that the high ZmMATE1 expression in root tips of Al tolerant maize lines is due to the presence of 3 nearly identical copies of ZmMATE1, and all three copies are functional. Al sensitive maize lines only have 1 copy of the ZmMATE1 gene in the root tip. For sub-objective 1B, we identified a novel mechanism of regulation of the sorghum Al tolerance protein, SbMATE. We identified a second protein, SbMBP (SbMATE binding protein), that binds very strongly to the SbMATE protein and regulates its function. We found that the binding of SbMBP to SbMATE blocks citrate transport and that SbMBP is an Al sensor and when it binds Al ions, it no longer binds to SbMATE, allowing the transport of citrate out of the root. This regulation of SbMATE ensures that unnecessary carbon loss from the root does not occur under non-Al toxic conditions, as citrate release from the root is a significant cost to the plant. In collaboration with Embrapa Maize and Sorghum, Brazil, we are using 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. For sub-objective 1C, we are improving our techniques for the imaging and reconstruction of root system architecture (RSA), which is important for acquisition of limiting nutrients like N, P and water, in 3 dimensions. Last year, our system to grow cereal roots in transparent gellan gum tubes was described that allows us to digitally image RSA in great detail and use our RootReader 3D software system to reconstruct the multiple images of the roots into a three dimensional model of the whole root system. The system was used successfully in the genetic mapping of rice RSA traits. For sorghum, we have developed a new and improved 3D growth system. The seedlings are now grown in glass cylinders in nutrient solution and not gellan gum. The 3D root system architecture is maintained in this hydroponic system by the series of plastic grids spaced at vertical intervals in the glass growth cylinder, allowing the roots to grow freely but as they grow through the mesh, the 3D architecture is maintained as the root systems are digitally imaged. This approach opens up many more avenues for our RSA research. For example, now we can readily modify the nutrient solution imposing P or N deficiency, or osmotic stress. Also, we can now image root systems of significantly older plants which allows us, for example, to study later developing root types such as sorghum crown roots that are important for water acquisition. Significant Activities that Support Special Target Populations: ARS researchers at Ithaca, NY have hosted two graduate students from the Department of Agricultural Sciences at Tennessee State University, an 1890 College with a tradition of educating under representative minorities in the sciences. The visitors were supported by an AFRI Strengthening Grant and worked with ARS scientists continuously, from June 18-August 13, 2012. They learned useful skills in protein chemistry and proteomics including multiplexed stable isotope labeling techniques, the operation high performance separations systems, the operation of state of the art mass spectrometers and the use of high throughput data analysis software. This experience enhanced their technical background, scientific knowledge, and problem solving skills. Furthermore the research carried out will be incorporated in there degree thesis. Additionally, draft versions of two manuscripts are being prepared for publication in peer reviewed journals concerning Al tolerance in tomato. ARS researchers in Ithaca also hosted a woman summer undergraduate intern. The student participated as part of a summer program between the USDA, Boyce Thompson Institute, and Cornell University funded by the National Science Foundation to involve under-represented minorities in plant science research. The student worked on a project with ARS scientists on maize sorghum aluminum tolerance over a 9 week period and then presented a 15 minute talk on the work at a Symposium held at the end of the 9 weeks. An ARS researcher is also serving as co-major professor of a female Latin-American PhD student in the Department of Plant Biology at Cornell University. The student has been awarded a minority fellowship from Cornell University and is working on her PhD research on rice adaption to soil-based abiotic stresses in the ARS lab. Accomplishments 01 Improved yields from sorghum grown on high acid soils. Acid soils make as much as 50% of the world�s soils, particularly in the tropics and subtropics where many developing countries are located. Also significant areas in the eastern and southern US have highly acidic soils. ARS researchers at the Robert W. Holley Center for Agriculture and Health at Ithaca, New York, discovered a unique protein in sorghum that allows sorghum to grow on acid soils. Knowing this, it will be possible to develop high yielding sorghum and other cereal crops to be grown on acid soils in developing countries including sub-Saharan Africa, Southeast As and South America, as well as on acid soils in the United States. 02 A second novel sorghum protein also improves sorghum yields on acid soil 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. Also significant areas in the eastern and southern US have highly acidic soils. ARS researchers at the Robert W. Holley Center for Agriculture an Health at Ithaca, New York, have discovered a second novel protein in sorghum that works in tandem with another sorghum protein they discovere allows sorghum to grow on acid soils. The discovery of these two protein can be used to more effectively develop high yielding sorghum and other cereal crops to be grown on acid soils in developing countries including sub-Saharan Africa, Southeast Asia and South America, as well as on acid soils in the United States. 03 New methods developed to identify an important modification to plant proteins. Proteins that control many processes in all organisms undergo specific modifications that are important for their function. One of the modifications is called phosphorylation, where a second protein inserts phosphate group onto the protein to be regulated. Phosphorylation is critical for the regulation on many proteins in all organisms, including important plant crop species. ARS researchers at the Robert W. Holley Center for Agriculture and Health at Ithaca, New York, have developed ne laboratory methods that will allow researchers to rapidly determine if a protein has been phosphorylated and where in the protein this has occurr This will help us understand how plant proteins that are involved in important crop traits, such as the ability to grow on acid soils, are regulated which in turn may help researchers enhance plant traits regulated by this phosphorylation process. 04 New methods developed to identify a second important modification to pla proteins. A second modification of plant proteins that is involved in t regulation of protein function is a process where sugar molecules are added to the protein, which is called glycosylation. ARS researchers at the Robert W. Holley Center for Agriculture and Health at Ithaca, New Yo have developed new laboratory methods that will allow researchers to rapidly determine if a protein has been glycosylated and to quantify the amount of these modified proteins in important plant crop species. This new technique will enable researchers to better understand the role of this protein modification process in important plant traits and possibly can be used to improve specific traits in plant species.
Impacts
(N/A)
Publications
- Desousa, S., Clark, R.T., Mendes, F., De Oliveira, A., De Vasconcelos, M., Parentoni, S.N., Kochian, L.V., Guimaraes, C.T., Magalhaes, J.V. 2012. A role for root morphology and related candidate genes in P acquisition efficiency in maize. Functional Plant Biology. DOI: org/10.1071/FP12022.
- Famoso, A., Zhao, K., Clark, R.T., Tung, C., Wright, M.H., Bustamante, C., Mccouch, S.R., Kochian, L.V. 2011. Genetic architecture of aluminum tolerance in rice (O. sativa) determined through genome-wide association analysis and QTL mapping. PLoS Genetics. 7(8):e1002221. DOI: 10. 137/journal.pgen.1002221.
- Liu, J., Luo, X., Shaff, J., Liang, C., Jia, X., Li, Z., Magalhaes, J., Kochian, L.V. 2012. A promoter swap strategy between the AtALMT and AtMATE genes increased arabidopsis aluminum resistance and improved carbon use efficiency for aluminum resistance. Plant Journal. 71(2):327-337.
- Li, L., Yang, Y., Xu, Q., Owsiany, K., Welsch, R., Chitchumroonchokchai, C. , Lu, S., Van Eck, J., Deng, X., Failla, M., Thannhauser, T.W. 2012. The Or gene enhances carotenoid accumulation and stability during post-harvest storage of potato tubers. Molecular Plant. 5:339-352.
- Zhou, X., Fei, Z., Thannhauser, T.W., Li, L. 2012. Transcriptome analysis of ectopic chloroplast development in green curd cauliflower (Brassica oleracea L. var. botrytis). Biomed Central (BMC) Plant Biology. 11:169.
- Yang, X., Yang, J., Pineros, M., Kochian, L.V., Zheng, S. 2011. Title A de novo synthesis citrate transporter VuMATE confers aluminum resistance in rice bean (vigna umbellata). Plant Cell and Environment. 34:2138-2148.
- Ligaba, A., Maron, L., Shaff, J.E., Kochian, L.V., Pineros, M. 2012. Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux. Plant, Cell & Environment. 35(7):1365-3040.
- Milner, M., Naoki, Y., Koyama, E., Jian, F., Kochian, L.V. 2012. Characterization of the high affinity Zn transporter from Noccaea caerulescens, NcZNT1, and dissection of its promoter for its role in Zn uptake and hyperaccumulation. New Phytologist. 195(1):113-123.
- Caniato, F., Guimaraes, C., Hamblin, M., Billot, C., Rami, J., Maciel, B.H. , Kochian, L.V., Liu, J., Garcia, A., Hash, C.T., Ramu, P., Mitchell, S., Kresovich, S., Oliveira, A., Avelar, G., Borem, A., Glaszmann, J., Schaffert, R.E., Magalhaes, J.V. 2011. The relationship between population structure and aluminum tolerance in cultivated sorghum. PloS One. 6(6) :e20830. DOI: 10.1371/journal.pone.0020830.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. 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. Significant Activities that Support Special Target Populations ARS researchers at Ithaca, NY, have hosted an Associate Professor and two of her graduate students from the Department of Agricultural Sciences at Tennessee State University, an 1890 College with a tradition of educating under representative minorities in the sciences. The visitors were supported by an AFRI Strengthening Grant and worked with ARS scientists continuously from July 18 through August 15, 2011. While in Ithaca, the visitors learned useful skills in protein chemistry and proteomics including multiplexed stable isotope labeling techniques, the operation high performance separations systems, the operation of state of the art mass spectrometers, and the use of high throughput data analysis software. This experience enhanced their technical background, scientific knowledge, and problem solving skills. Furthermore, the research carried out by the two students will be incorporated in their degree thesis. Additionally, draft versions of two manuscripts are being prepared for publication in peer reviewed journals concerning drought and salt tolerance in tomato. ARS researchers in Ithaca also hosted an African-American woman summer undergraduate intern. The student participated as part of a summer program between the USDA, Boyce Thompson Institute, and Cornell University funded by the National Science Foundation to involve under- represented minorities in plant science research. The student worked on a project with ARS scientists on sorghum aluminum tolerance over a 9 week period and then presented a 15 minute talk on the work at a Symposium held at the end of the 9 weeks. Accomplishments 01 Over 20% of the US land area and up to 50% of the world�s arable lands a highly acidic. On these acid soils, aluminum (Al) ions are dissolved int the soil solution which are toxic to and damage plant roots, thus greatl reducing crop vigor and yields. Because of the importance of this proble 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 abou the genetics of rice Al tolerance. ARS researchers at the Robert W. Holl Center for Agriculture & Health at Ithaca, NY, used novel statistical genetic approaches in a large rice diversity panel to identify new regio 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. 02 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 countri 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 b 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 n 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 importan staple food crop on acid soils prevalent in sub-Saharan Africa. 03 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 pla root systems to crop traits such as nutrient and water acquisition. Researchers are discovering that the way the plant determines where in t soil profile its roots grow makes a difference; for example, in acquirin 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 Agricultur & Health at Ithaca, NY, developed a system to grow root systems of cerea 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 limitin conditions. Subsequently, this information will be used to identify gen 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.
Impacts
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Publications
- 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.
- 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.
- 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.
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