2008 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.
This report documents work conducted for CRIS 1907-21000-024-00D. For the sorghum aluminum (Al) tolerance research, we continued to characterize the sorghum Al tolerance gene we cloned last year, AltSB, which encodes a MATE transporter mediating Al-activated root citrate exudation that is central to sorghum Al tolerance. We developed near isogenic lines (NIL) that are genetically identical except for the region around AltSB, where each NIL harbors a different version of AltSB generated from backcrossing different sorghum lines with the same parent. Using these lines, we are identifying the most effective versions of this gene to be used in molecular breeding programs. We also obtained clear evidence for the existence of several new sorghum Al tolerance genes that are functioning in specific sorghum lines. Furthermore, working with the NILs we have found strong evidence that these novel tolerance genes interact with AltSB to facilitate maximal AltSB gene expression and tolerance.
Al tolerance in maize is a complex involving multiple genes, unlike sorghum where a single tolerance gene is usually involved. We have greatly extended earlier quantitative trait loci (QTL) mapping work using a population from a cross between tolerant and sensitive tropical maize lines. Using this mapping population, six Al tolerance QTL – or 6 different regions of the maize genome where Al tolerance genes reside - were identified. The QTL on chromosomes 5 and 6 explain a majority of the variation in tolerance. Also, using maize gene chips, we have conducted a detailed analysis of root tip (the site of Al tolerance) gene expression under Al stress with the Al-tolerant and sensitive parents of the mapping population. It was found that several maize members of the MATE gene family exhibited much higher expression in the root tips of the tolerant line compared with the sensitive line. The most dramatic differences in expression are for the genes we have designated ZmMATE1 and 2. Genetic mapping of ZmMATE1 confirmed it is located at the major Al tolerance QTL on chromosome 6, while ZmMATE2 maps to the major Al tolerance QTL on chromosome 5. Hence these are strong candidates for the first identified maize Al tolerance genes.
For our work on proteomic analysis, which involves the use of novel liquid chromatograph (LC) protein separation techniques combined with new mass spectrometry methods (MS/MS) to analyze and identify plant proteins, we have made significant progress through the application of multiple reaction monitoring (MRM) methods to our existing LC-MS/MS techniques. These can be used to sensitively and accurately verify changes in protein expression, which has been quite difficult to achieve previously. Furthermore, the MRM approaches under development can be used to directly test hypotheses concerning the differential expression of proteins generated by non-proteomic methods such as from gene chips or genetics-based studies.
This work fits into Component I: Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement, and Component 2. Biological Processes that Improve Crop Productivity and Quality, of the NP302 Action Plan.
Two different Al tolerance genes work together in the model plant, Arabidopsis. We had previously shown that the ALMT1 gene, which stands for aluminum (Al) activated malate transporter, is the major Al tolerance gene in the model plant species, Arabidopsis thaliana. Working with mutant plants where the ALMT1 gene was rendered non-functional, we identified a second Al tolerance gene that is closely related to our sorghum Al tolerance gene. This gene is not related to ALMT1 and encodes an Al-activated citrate efflux transporter. This is the first demonstration that two different Al tolerance genes function together to confer the full range of Al tolerance in a plant species. This research will help facilitate the development of crop plants resistant to Al toxicity and addresses 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.
Methods to reduce false discovery rates of proteins through direct hypotheses testing. We have developed new methodologies to eliminate or reduce the rate of mis-identification of proteins isolated and identified via combined liquid chromatograph-mass spectrometry methods. This is based on a procedure where proteins are separated based on the pH value where they have no net electric charge (peptide isoelectric point filtering), and the use of identification hypotheses testing via chemical modification of specific structures within the protein. This accomplishment allows for much more accurate identification of specific proteins isolated from a mixture of thousands of proteins in a plant tissue extract. This work addresses 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.
Identification of a novel protein that interacts with our sorghum Al tolerance protein, AltSB. Acid soils comprise large areas of land in the US and throughout the world, and 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 characterizing the molecular and physiological basis for plant tolerance to Al toxicity, in order to improve crops for agriculture on acid soils. In studies aimed at characterizing a major sorghum Al tolerance gene we have identified which encodes a membrane transporter that pumps Al-detoxifying citric acid out of root cells, we searched for smaller proteins that bind to this citrate transporter. A unique metal-binding protein was identified that binds very tightly to the citrate transporter protein. We think this smaller protein “senses” Al ions and then binds to the tolerance protein, regulating its function. This work addresses 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 unit hosted an African-American undergraduate research intern, who will be a junior at Xavier University, a Historically Black College. Individual was supported by a National Science Foundation Research Experiences for Undergraduates (NSF-REU) grant and worked in the unit for the 2008 summer (6/2/08 to 8/8/08). 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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Jones, D.L., Blancaflor, E., Kochian, L.V., Gilroy, S. 2008. Spatial Coordination of Aluminum Uptake, Production of Reactive Oxygen Species, Callose Production and Wall Rigidification in Maize Roots. Plant Cell and Environment. 29:1309-1318.
Klein, M., Sekimoto, H., Milner, M., Kochian, L.V. 2008. Investigation of Heavy Metal Hyperaccumulation at the Cellular Level: Development and Characterization of Thlaspi caerulescens Suspension Cell Lines. Plant Physiology. 147:2006-2016.
Maron, L.G., Matias, K., Mao, C., Menossi, M., Kochian, L.V. 2008. Transcriptional profiling of Al toxicity and tolerance responses in maize roots. New Phytologist. 179:116-128.
Parameswaran, A., Leitenmaier, B., Yang, M., Kroneck, P., Welte, W., Lutz, G., Papoyan, A., Kochian, L.V., Kupper, H. 2008. A native Zn/Cd transporting P1B ATPase from natural overexpression in a hyperaccumulator plant reveals post-translational processing. Biochemical and Biophysical Research Communications. 363:51-56.
Pineros, M., Kochian, L.V. 2008. Novel properties of the wheat aluminum tolerance organic acid transporter (TaALMT1) revealed by electrophysiological characterization in Xenopus oocytes: Functional and structural implications. Plant Physiology. 147:2131-2146.
Klein, M., Papoyan, A., Kochian, L.V. 2006. Phytoremediation. In: Mcgraw-Hill Yearbook of Science and Technology. p. 115-125.
Li, L., Lyi, S.M., Zhou, X., Kochian, L.V. 2007. Biochemical and molecular characterization of the homocysteine s-methyltransferase from broccoli (brassica oleracea var. italica). Phytochemistry. 68:1112-1119.
Pineros, M., Cancado, G., Maron, L., Lyi, S., Menossi, M., Kochian, L.V. 2008. Not all ALMT1-type transporters mediate aluminum-activated organic acid responses: The case of AmALMT1-an anion selective transporter. Plant Journal. 53:352-367.
Kobayashi, Y., Hoekenga, O., Ito, H., Nakashima, M., Saito, S., Shaff, J., Maron, L., Pineros, M., Kochian, L.V., Koyama, H. 2008. Characterization of AtALMT1 expression in aluminum inducible malate release and its role for rhizotoxic stress in Arabidopsis. Plant Physiology. 145:843-852.
Yang, Y., Howe, K.J., Zhang, S., Wilson, D., Moser, F., Irwin, D., Thannhauser, T.W. 2007. A comparison of nlc-esi-ms/ms and nlc-maldi-ms/ms for gelc-based protein identification and itraq-based shotgun quantitative proteomics. Journal of Biomolecular Techniques. 18:226-237.
Yang, X., Thannhauser, T.W., Burrows, M.E., Cox Foster, D., Gildow, F., Gray, S.M. 2008. Coupling genetics and proteomics to identify aphid proteins associated with vector-specific transmission of Polerovirus (Luteoviridae). Journal of Virology. 82:291-299.