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United States Department of Agriculture

Agricultural Research Service

2010 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
For the sorghum aluminum (Al) tolerance research, we have been identifying Al tolerance genes to exploit in order to increase sorghum yields on the acidic, Al toxic soils that reduce yields on 30-50% of the world’s lands, including many developing countries. In collaboration with researchers from Embrapa Maize and Sorghum in Brazil, we previously identified the first Al tolerance gene in sorghum, SbMATE, which encodes a root transporter that pumps the organic acid, citric acid, into the soil to detoxify the toxic Al ions. We now have identified a second protein from the cytochrome family of metal-binding proteins that interacts very strongly with the SbMATE protein and appears to regulate its function. Our working hypothesis is that this new protein binds to SbMATE and prevents the transport of citric acid in the absence of Al ions. When the protein binds Al ions, it undergoes a conformational change allowing citric acid to be transported, thus ensuring that significant 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 & investigating the possibility to increase the productivity of biofuel sorghum grown on acid soils prevalent in the southeastern U.S. We identified the first maize al tolerance gene, ZmMATE1, based on its similarity to the sorghum SbMATE gene. We showed that ZmMATE1 encodes a root citric acid transporter that is expressed much more highly in root tips of Al tolerant maize and Al exposure increases this already high expression. These findings open up new avenues for improving the acid soil tolerance of maize via molecular breeding.

Rice is the most Al tolerant cereal and most important food crop in the world, yet little is known about its high Al tolerance. We found that rice uses novel Al tolerance mechanisms not based on root organic acid release. We then conducted “association genetics” analysis of rice Al tolerance which involves phenotyping of a panel of 400 different rice lines for Al tolerance. This diversity panel has been genotyped with over 44,000 unique molecular markers. Association genetics involved using a novel statistical program to identify molecular markers that associate with rice Al tolerance. Using this approach, we quickly identified a number of novel regions of the rice genome where Al tolerance genes reside and are in the process of evaluating candidate Al tolerance genes.

Proteomics analysis of Al tolerance was conducted using tomato plants as a model crop system. This analysis identified 49 proteins isolated from tomato roots that undergo a statistically significant change in expression upon exposure to toxic Al ions. We also identified a set of tomato proteins isolated from young shoot tissue of tomatoes that suggest that antioxidant and detoxification as well as sugar metabolism pathways are enhanced under Al stress, while the photosynthetic and carbon fixation machinery are suppressed. These findings indicate that root organic acid transport & shoot carbon metabolic pathways are coordinately regulated during the operation of A1 tolerance mechanisms.

1. Identification of a novel protein that regulates the sorghum aluminum tolerance protein. 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 crop production by damaging plant roots. ARS scientists at the Robert W. Holley Center for Agriculture & Health in Ithaca, NY have discovered the primary Al tolerance protein in sorghum (Sorghum bicolor) that we named SbMATE. SbMATE pumps organic acids out of the root tip into the soil, where the organic acids detoxify Al, allowing the root to grow. Here we have identified a second sorghum protein that binds very tightly to the organic acid pump protein. Our current findings are consistent with the hypothesis that this second protein binds to and regulates the function and efficiency (organic acid transport activity) of the primary Al tolerance protein. 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 African staple food crop on acid soils prevalent in sub-Saharan Africa.

2. Regulation of expression of the SBMATE gene and the protein it encodes at the tissue and cell levels. Aluminum (Al)-activated exudation of organic acid (OA) anions from root apices is the best documented and characterized plant Al tolerance mechanism. In sorghum (Sorghum bicolor), Al-activated citric acid exudation from root apices of the Al-tolerant sorghum lines is controlled by SbMATE, which encodes a plasma-membrane-localized citrate transporter that belongs to the multidrug and toxic compound extrusion (MATE) family. SbMATE is expressed primarily in the root apices of Al tolerant lines and is induced by Al, ultimately being responsible for the observed Al-activated root citrate exudation. However, SbMATE transcript and protein abundance at tissue and cellular levels is still not known. To address this question, ARS scientists at the Robert W. Holley Center for Agriculture & Health in Ithaca, NY used microscopic techniques with a labeled antibody that binds to SbMATE protein to determine the cellular location of SbMATE. For SbMATE gene expression localization, we used a laser to microdissect a few cells away from a specific region of the sorghum root and then used molecular techniques to quantify SbMATE gene expression in those cells. We found that SbMATE gene and protein expression is localized to a very discrete region of the root which we also determined was the root region most susceptible to Al stress. The SbMATE gene and protein are mainly expressed in the outer layers of the root (the root epidermal cells) and specifically in a discrete, approximately 2 mm wide zone between the root regions of cell division and cell elongation (labeled the distal transition zone). These findings show how elegantly this Al tolerance process is regulated to ensure that the most susceptible root region is protected from Al stress and at the same time the energetic cost to the plant with regards to carbon loss is minimized.

3. Molecular analysis of two Arabidopsis Al tolerance genes, AtMATE and AtALMT. In the model plant species Arabidopsis thaliana, Al tolerance is based on a high level of Al-induced root malic acid exudation and a lower level of Al-induced root citric acid exudation. These two processes are mediated by two organic acid transporters, AtALMT1 which is the malic acid transporter, and AtMATE, the root citric acid transporter. For this molecular analysis of expression of these two genes, ARS researchers at Ithaca, NY, first generated a double mutant where both the AtALMT1 and AtMATE genes were knocked out, so that the mutant plants lack both Al-activated malic and citric acid exudation. We then made synthetic genes where we swapped the gene promoters (the region of the gene that drives gene expression) for each gene so that we created the AtMATE gene driven by the AtALMT1 promoter and the AtALMT1 gene driven by the AtMATE promoter. We then expressed each synthetic gene independently in transgenic double mutant Arabidopsis plants lacking the normal AtMATE and AtALMT1 genes, and quantified their Al tolerance compared with normal plants and the double mutant plants not expressing either gene. We found that the gene where the AtALMT1 promoter drives expression of the AtMATE gene conferred a large increase in Al tolerance, much more than transgenic plants expressing the AtALMT1 gene driven by the AtMATE promoter. These findings indicate that the AtALMT1 promoter is much more effective in directing the expression of Al tolerance genes and that citric acid exudation mediated by AtMATE has a bigger effect on Al tolerance than malic acid exudation. These findings will help direct biotechnological approaches for improving crop plant Al tolerance via the generation of transgenic plants expressing engineered plant Al tolerance genes.

4. Development of more effective and efficient system for separating and identifying plant proteins. ARS researchers at Ithaca, NY, have developed a high throughput, high resolution 2-dimensional peptide separation system. There is a growing need to be able to identify and quantify proteins whose expression changes in response to both biotic and abiotic stress. The system developed is more easily automated and improves the resolution achieved by combining two compatible separation strategies that are carried out under substantially different solution conditions. It allows for the identification of 50% more proteins than the methods used previously. The new method will help us to identify more proteins from complex samples and improve our ability to dissect the complex molecular pathways that control how plants interact with their environments.

5. Development of improved proteomic platform to enhance ability to identify regulatory proteins. There is a growing awareness of the importance of protein phosphorylation in the regulation of important biological processers. Identifying phosphorylated proteins and finding their sites of phosphorylation is difficult because they are typically low abundance proteins created by modification reactions that leave the modification sites only partially occupied (non-stoichiometric). Computational strategies are often utilized to predict the location of phosphorylation sites, however there is a need for improved empirical methods to validate computational predictions and to provide for new hypotheses. To find and identify phosphorylated peptides in a background of non-phosphorylated peptides requires a method of enrichment. ARS researchers at Ithaca, NY, have developed a workflow incorporating Metal Oxide Affinity Chromatography to enrich for and identify phosphopeptides in digests of protein extracts from plant tissues that will aid in the identification of the regulatory proteins involved in important biological processes including aluminum tolerance.

5.Significant Activities that Support Special Target Populations
Hosted an Assistant Professor and two of her graduate students from Tennessee State University, an 1890 College with a tradition of educating under-represented minorities in the sciences. The visitors were supported by an AFRI Strengthening Grant and worked with ARS scientists continuously, from May 13-August 4, 2010. They learned useful skills in protein chemistry and proteomics including 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.

Hosted two African graduate students from Moi University in Kenya who spent 8 months at the Holley Center in Ithaca, NY, learning physiological and molecular approaches for the study of maize and sorghum Al tolerance. This involved phenotyping hydroponically grown maize and sorghum plants for Al tolerance, quantifying root organic acid exudation, cloning sorghum and maize Al tolerance genes (SbMATE and ZmMATE1) and learning quantitative real-time PCR to quantify SbMATE and ZmMATE1 gene expression. All of this research was conducted on maize and sorghum diversity panels brought to the USDA lab from Kenya, consisting of more than 100 sorghum and maize lines adapted to Kenya and used for Kenyan maize and sorghum breeding programs. From this research, draft versions of two manuscripts – one on maize Al tolerance and the other on sorghum Al tolerance, were written by the two students and are being edited for submission to peer-reviewed journals.

Review Publications
Li, L., Zhou, X., Yuan, Y., Yang, Y., Rutzke, M., Thannhauser, T.W., Kochian, L.V. 2009. Involvement of a broccoli COQ5 methyltransferase in the production of volatile selenium compounds. Plant Physiology. 151:528-540.

Kuepper, H., Kochian, L.V. 2010. Transcriptional regulation of metal transport genes and mineral nutrition during acclimation to cadium and zinc in the Cd/Zn hyperaccumulator, Thlaspi caerulescens (Ganges population). New Phytologist. 185(1):114-129.

Ligaba, A., Kochian, L.V., Pineros, M. 2009. Phosphorylation at S384 regulates the activity of the TaALMT1 malate transporter that underlies aluminum resistance in wheat. Plant Journal. 60:411-423.

Shaff, J., Schultz, B., Craft, E.J., Clark, R., Kochian, L.V. 2010. GEOCHEM-EZ: a chemical speciation program with greater power and flexibility. Plant and Soil Journal. 330:207-214.

Wang, Y., Mosebach, C.M., Kibbe, A.S., Ryhal, M.K., Jones, A.D., Palmer, J.A., Kochian, L.V. 2009. Generation of Arabidopsis mutants by heterologous expression of a full length cDNA library from tomato fruits. Plant Molecular Biology Reporter. 27:454-461.

Maron, L., Guimaraes, C., Pineros, M., Magalhaes, J., Pleiman, J., Mao, C., Kochian, L.V. 2010. Two functionally distinct members of the MATE (multidrug and toxic compound extrusion) family of transporters potentially underlie two major Al tolerance QTL in maize. Plant Journal. 61:728-740.

Yang, Y., Thannhauser, T.W., Li, L., Zhang, S. 2007. Development of an integrated approach for evaluation of 2-D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2-D gel/MALDI workflow. Electrophoresis. 28:2080-2094.

Macarisin, D., Wisniewski, M.E., Bassett, C.L., Thannhauser, T.W. 2009. Proteomic analysis of B-aminobutyric acid priming and aba-induction of drought resistance in crabapple (Malus pumila): effect on general metabolism, the phenylpropanoid pathway and cell wall enzymes. Plant Cell and Environment. 32: 1612-1631.

Suping, Z., Zhou, S., Fish, T., Thannhauser, T.W. 2009. Salt Induced and Salt Suppressed Proteins in Tomato Leaves. Journal of the American Society for Horticultural Science. 134(2):289-294.

Cilia, M., Fish, T., Yang, X., Mclaughlin, M., Thannhauser, T.W., Gray, S.M. 2009. A Comparison of Protein Extraction Methods Suitable for Gel-Based Proteomics Studies of Aphid Proteins. Journal of Biomolecular Techniques. 20(4):201-215.

Alos, E., Roca, M., Iglesias, D., Minguez-Mosquera, M., Borges-Damasceno, C., Thannhauser, T.W., Campbell-Rose, J., Talon, M., Cercos, M. 2008. An evaluation of the basis and consequences of a stay-green mutation in the navel negra (nan) citrus mutant using transcriptomic and proteomic profiling and metabolite analysis. Journal of Plant Physiology. 147:1300-1315.

Filiatrault, M.J., Stodghill, P., Bronstein, P., Moll, S., Lindeberg, M., Grills, G., Schweitzer, P., Wang, W., Schroth, G., Luo, S., Khrebtukova, I., Thannhauser, T.W., Yang, Y., Butcher, B.G., Cartinhour, S.W., Schneider, D.J. 2010. Transcriptome analysis of Pseudomonas syringae identifies new genes, ncRNAs, and antisense activity. Journal of Bacteriology. 192(9):2359-2372.

Last Modified: 9/2/2015
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