2013 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.
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.
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.
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.
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.
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.
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.
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.
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.