2009 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.
For the sorghum aluminum (Al) tolerance research, we have been using the information from our basic research to develop sorghum lines that have higher yields on the acidic, Al toxic soils that limit agriculture on between 30-50% of the world’s lands, including developing countries in Africa, Asia and South America. Building upon our previous discovery of the first Al tolerance gene in sorghum, SbMATE, we analyzed its sequence and expression in a sorghum diversity panel of 500 sorghum lines chosen to represent most of the sorghum diversity worldwide. Sequence analysis of the SbMATE from the most Al tolerant sorghum lines allowed us to identify several molecular markers that are diagnostic for the most Al tolerant versions of this gene. We are working with sorghum breeders in Niger and Kenya to screen their local germplasm with this marker to identify the most effective SbMATE genes, and this germplasm will be used for sorghum breeding to improve sorghum productivity on acid soils in both western and eastern Africa. Similar approaches can also be sued to improve productivity of sweet sorghums grown for biofuels on the acid soils prevalent in the southeastern U.S.
We have identified two Al tolerance genes (including SbMATE) and both are organic acid transporters that pump Al detoxifying organic acids from the root tip into the soil. We express these transporters in frog eggs, which is an expression system for the study of transporter genes. Using computer analysis to make predictions about the protein structure of these transporters, we identify regions of the protein that are predicted to be important for the transport properties associated with Al tolerance. We then modify these regions using recombinant DNA techniques and investigate the resulting changes in their transport properties. The goal of this research is to obtain the fundamental information needed to “engineer” these Al tolerance proteins to improve their efficacy in conferring Al tolerance in crop species.
Rice is the most Al tolerant of the cereals and the most important food crop in the world. Yet little is known about the mechanisms conferring its high degree of Al tolerance. We assembled a diversity panel for rice capturing most of the genetic diversity in rice worldwide, and are analyzing this panel for genetic variation in Al tolerance. To improve our ability to more accurately phenotype individual plants for Al tolerance, we developed new root imaging techniques (see Accomplishment.
1)that greatly facilitated this analysis. We have identified several novel regions of the rice genome which harbor Al tolerance genes and are in the process of cloning these genes. We integrated this molecular genetic research with a physiological analysis of rice Al tolerance and discovered that unlike sorghum, maize or wheat, the very high Al tolerance in rice does not depend on organic acid release from the roots. Thus it is clear that rice uses novel Al tolerance mechanisms and identifying the genes underlying this tolerance will greatly expand our “molecular toolbox” for improving crop Al tolerance.
Discovering how aluminum tolerance proteins are regulated. Acid soils make up between 30 and 50% of the world’s soils, particularly in the tropics and subtropics where many developing countries are located. On acid soils aluminum (al) toxicity limits agriculture by inhibiting and damaging roots. We have discovered several Al tolerance proteins that are transporters that pump organic acids out of the root tip into the soil, where they detoxify Al. Many proteins are regulated by a process known as phosphorylation, where a specific enzyme, a kinase enzymatically places a phosphate group on an amino acid in the protein. For these organic acid transporters that confer Al tolerance, we have been using an integrated computational and molecular biology approach to study the relationship between protein structure and organic acid transport. This has enabled us to identify a specific group of 3 amino acids that is phosphorylated in these organic acid transporters, which then greatly enhances their ability to pump organic acids. We hope to use this information to begin to engineer more effective Al tolerance proteins for cereal crop species.
High throughput root imaging techniques developed. There is a growing understanding that root architecture, the placement of specific roots in an entire root system in relation to other specific roots and in relation to different positions in the soil, is an important trait for crop productivity. This is particularly true for crop plants experiencing abiotic stresses such as limiting amounts of water, phosphorous and/or toxic levels of metals such as aluminum. To begin to quantify whole root system architecture for molecular genetic analyses, we developed flexible growth and imaging systems and software tools that have allowed us to image and analyze root systems both in 2 and 3 dimensions to study root system characteristics for large numbers of plants. Using this system, we were able to quickly phenotype over 10,000 rice seedlings in a 500 line rice diversity panel for Al tolerance, which has rapidly advanced our ability to identify molecular determinants for rice Al tolerance. These new techniques will enable us to begin to determine the role of genetically-based differences in root architecture in important plant traits such as drought tolerance and the ability to acquire limiting nutrients such as phosphorous.
5.Significant Activities that Support Special Target Populations
Hosted a Hispanic undergraduate research intern from Fresno State University. Individual was supported by a National Science Foundation Research Experiences for Undergraduates (NSF-REU) grant and worked with ARS scientists for the 2009 summer. He learned useful molecular laboratory skills, and gained experience in critical reading of the scientific literature which enhanced his science knowledge and problem solving skills.
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Milner, M., Kochian, L.V. 2008. Investigating Heavy-metal Hyperaccumulation using Thlaspi caerulescens as a Model System. Annals Of Botany. 102(1):3-13.
Liu, J., Magalhaes, J., Shaff, J., Kochian, L.V. 2009. Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance. Plant Journal. 57(3):389-399.
Sooksa-Nguan, T., Yakubov, B., Kozlovskyy, V.I., Barcume, C.M., Thannhauser, T.W., Rutzke, M.A., Hart, J.J., Kochian, L.V., Vatamaniuk, O.K. 2009. Drosophila ABC Transporter DmHMT-1 Confers Tolerance to Cadmium. Journal of Biological Chemistry. 284:354-362.
Jin, F., Frohman, C., Glahn, R.P., Thannhauser, T.W., Welch, R.M. 2009. Effects of Ascorbic Acid, Phytic Acid and Tannic Acid on Iron Bioavailability from Reconstituted Ferritin Measured by an In Vitro Digestion/Caco-2 Cell Model. British Journal of Nutrition. 101(7):312-316.
Xiang, B., Yang, X., Thannhauser, T.W. 2009. Protein N- and C-Termini Identification Using Mass Spectrometry and Isotopic Labeling. Journal of Rapid Communications in Mass Spectroscopy. 23:2102-2106.
Zhu, J., Zhang, X., Roneker, C.A., Mcclung, J.P., Zhang, S., Thannhauser, T.W., Ripoll, D.R., Sun, Q., Lei, X. 2008. ROLE OF COPPER,ZINC-SUPEROXIDE DISMUTASE IN CATALYZING NITROTYROSINE FORMATION IN MURINE LIVER. Journal of Free Radical Biology and Medicine. 45:611-618.