Progress 10/01/08 to 09/30/09
OUTPUTS: For research on TaALMT1, the malate efflux transporter which underlies wheat aluminum (Al) tolerance, we have conducted the following: 1) Functional modulation of TaALMT1 transport activity by ligand (i.e. Al3+) binding and/or phosphorylation was evaluated via Two Electrode Voltage Clamp (TEVC) in Xenopus oocytes expressing this transporter. Previous pharmacological results established the role of phosphorylation in the modulation of TaALMT1 transport activity. We have evaluated eight different single point mutations modifying six predicted phosphorylation sites. Two of these residue substitutions in the hydrophilic C-terminus resulted in altered transport. 2) Functional analysis of different chimeric-proteins where the where the hydrophilic C- terminus region has been truncated along several places as well as chimeric proteins where the pore forming region (N-terminus) domains and hydrophilic region (C-terminus) domains of three ALMT orthologs have been swapped among each other. These results have provided insight into the role of the different protein domains. Regarding work on the sorghum Al tolerance gene/protein SbMATE that is a root citrate efflux transporter, we have done the following: 1) Continued research on the expression of SbMATE protein and gene at the tissue and cellular levels by immunolocalization and via in situ mRNA localization. 2) Began to characterize two cytochrome b5-like proteins in sorghum identified via split ubiquitin yeast-2-hybrid as strong candidates for the SbMATE interacting proteins. 3) Conducted electrophysiological experiments to demonstrate that SbMATE functions as a citrate transporter and is inhibited by Al in oocytes, , which is different from the observation in plants where the root citrate exudation is activated by Al. 4) Expressed GST-cytochrome b5 like recombinant proteins in E. coli and purified the recombinant proteins through affinity chromatograph and began to study their ability tobind al and other metals. 5) Using mass spectrometry to test the ability of the cyt b-5 proteins to bind SbMATE before and after Al binding. 6) Studying the role of closest related cyt b5 protein homologs in Arabidopsis via T-DNA knockouts. Our findings have been shared with the research community via the following presentations (selected talks shown due to space limitations): 1. Keynote talk "Elucidating the molecular basis of crop aluminum tolerance using sorghum as a model system", COMBIO 2008 Conference, Canberra, Australia, 2008. 2. Keynote opening address "Molecular and physiological basis for crop aluminum resistance", 8th International Symposium on Plant-Soil Interactions at Low pH, Guangzhou, China 2009. 3. Plenary address "Adaptive strategies for plant response to toxic metals in the soil", XVI International Plant Nutrition Colloquium, Sacramento, CA 2009. 4. Plenary talk "Molecular and genetic dissection of aluminum tolerance in maize and sorghum", Generation Challenge Program Annual Research Meeting, Bamako, Mali 2009. 5. Invited talk "Elucidating the molecular basis for cereal aluminum tolerance", Abiotic Stress Symposium, International Plant Molecular Biology Congress, St. Louis, MO 2009. PARTICIPANTS: Dr. Miguel Pineros is an ARS Research Associate in Dr. Kochian's laboratory and is leading the project. Dr. Ayalew Ligaba is the Postdoctoral Associate hired under this grant, and is currently working on the several areas of the project. Dr. Ligaba has been implementing electrophysiological techniques (e.g., two electrode voltage clamp) characterizing the functionality of the various chimeras, mutations, and truncations of the ALMT-family of proteins. Dr. Jiping Liu, ARS SY, Kochian Lab. Dr. Liu is the lead scientist for the work on the cytochrome protein that binds tightly to and possibly regulates SbMATE. He also was the lead on the work on the homolog of SbMATE in Arabidopsis, which was published in the Plant Journal in 2009. Lyza Maron is a BTI postdoctoral associate in the Kochian Lab, who was the lead on the cloning and characterization of ZmMATE1, the maize homolog of SbMATE that is the first maize Al tolerance gene cloned. She is first author on the paper in press in Plant Journal. Miguel Pineros is an ARS Research Associate in the Kochian lab who leads the work on expression of SbMATE, ZmMATE1 and other related transporters in Xenopus oocytes and studies their transport function. Jurandir Magalhaes and Claudia Guimaraes are collaborators at Embrapa Maize and Sorghum in Brazil who have collaborated for many years with the Kochian lab on this research. The cloning of SbMATE began when Dr. Magalhaes was a PhD student with Dr. Kochian and has continued after he took at job with Embrapa. he has continued to collaborate on the sorghum Al tolerance work and he was first author on the 2007 Nature Genetics paper. His colleague, Dr. Guimaraes, has collaborated on the ZmMATE1 work and is a co-author in the Plant Journal paper in press. Thomas Matonyei and Reuben Kiboiare and Kiboi are MS students in the lab of Dr. Sam Gudu, Moi University, Kenya. They worked for 1 year in the Kochian lab on some of this grant's research learning molecular and genetic techniques while screening African lines of sorghum and maize for Al tolerance and root organic acid exudation. They also cloned SbMATE from sorghum and its homolog in maize and investigated the expression of these genes via real-time PCR in a number of the sorghum and maize lines they brought from Kenya. TARGET AUDIENCES: This work will have direct implications for farmers whose crop yields are reduced by Al toxicity on acid soils. Since acid soils comprise many of the soils in the tropics and subtropics where developing countries are located, Al toxicity limits crop yields in those very countries where food security is most tenuous. The information on sorghum and maize Al tolerance generated from the basic research funded by this grant is now being translated into real-world agricultural improvement via collaborations between the Kochian lab and sorghum and maize breeders in West and East Africa. We are collaborating with Sam Gudu of Moi University, Kenya, Soumana Souley of INRAN in Niger, Eva Weltzien of ICRISAT in Mali, and Jurandir Magalhaes and Robert Schaffert of Embrapa Maize and Sorghum to use this basic information regarding SbMATE and ZmMATE1 to screen African maize and sorghum germplasm for strong alleles of these genes. We have identified easy to score markers for very Al tolerant alleles of SbMATE and ZmMATE1 and are genotyping African maize and sorghum germplasm for lines harboring these alleles. Subsequently these lines are being phenotyped on acid soils in the field in Africa to verify the trait, and then are introduced into sorghum and maize breeding programs in Sub-Saharan Africa. PROJECT MODIFICATIONS: Not relevant to this project.
With regards to TaALMT1, the wheat root malate efflux transporter, we found its transport properties are highly dependent/enhanced by the binding of Al3+ to the protein, even though the protein can mediate malate release without Al3+. This Al enhancement of malate transport is due to increases in ALMT1's anion permeability. We are investigating the structural features of the protein important for anion selectivity and Al activation by systematically altering structural motifs/domains and examining the effect of these alterations on the function of the protein. This type of analysis has shown that the first half (N-terminus containing the hydrophobic regions) of the protein forms the pore region of the protein that can mediate ion transport even in the absence of the C-terminus hydrophilic portion of the protein. However, the presence of the C-terminus (hydrophilic regions) is required for Al activation of malate efflux. We also have found that the functional TaALMT1 protein assembles as an aggregate of several TaALMT1 subunits (i.e. multimer). We have established that the transport activity of TaALMT1 can be regulated by alternate secondary messenger cascades. Pharmacological studies of TaALMT1 and TaALMT1 containing single point mutations have indicated that protein phophorylation events taking place at the C-terminus region are a fundamental requirement for TaALMT1 activity. The understanding of the structural features underlying the functional characteristics of ALMT-type transporters should enable the design of similar transporters with enhanced functional (e.g. substrate and regulatory) properties, ultimately improving their ability to confer Al tolerance in crop plants. We also have shown that SbMATE, the major sorghum Al tolerance gene that encodes an Al-activated citrate transporter in sorghum, is regulated in part by binding other proteins. We have identified two cyt b5 like proteins as strong candidates for SbMATE interacting components. W found that when expressed in Xenopus oocytes, the SbMATE-mediated citrate exudation is independent of Al, suggesting that there is an Al-dependent negative regulator(s) in plants that suppresses the SbMATE citrate transporter in the absence of Al stress. The negative regulator of SbMATE could also function as an Al sensor: in the presence of Al stress, the regulator binds to Al and changes its own conformations, which could lead to inability to of the negative suppressor to suppress SbMATE-mediated citrate transport from cells. As the SbMATE-interacting cytochrome b5 like proteins, identified from our yeast-two hybrid screens, belong to a metal-binding protein family, they could be strong candidates for the negative regulators of SbMATE as well as an Al sensor. We have expressed and purified the sorghum cytochrome b5 like recombinant proteins from E. coli and are currently testing if these proteins bind Al3+ and if they do, does this confer regulation of citrate transport by SbMATE. This research could lead to the first identification of a sensor for Al stress in plants and a better understanding of the molecular and cellular mechanisms that underlie how plants tolerate/avoid Al stress.
- Clark, Kochian LV. 2009. Investigating whole root systems: Advances in root quantification tools and techniques. Proceedings of the 7th International Symposium on Plant-Soil Interactions at Low pH, Guangzhou, China, pp. 143-144.
- Guimaraes CT, Magalhaes JV, Jardim SN, Almeida RV, Maron L, Parentoni SN, Alves VMC, Lana UGP, Gama EEG, Hoekenga O, Paiva E, Kochian LV. 2009. QTL and selection mapping for Al tolerance in tropical maize. Proceedings of the 7th International Symposium on Plant-Soil Interactions at Low pH, Guangzhou, China, pp. 147-148.
- Koyama H, Iuchi S, Hoekenga O, Kobayashi Y, Sawaki Y, Kobayashi Y, Kochian LV, Kobayashi W. 2009. Signal transduction pathway of the Al responsive malate release in Arabidopsis. Proceedings of the 7th International Symposium on Plant-Soil Interactions at Low pH, Guangzhou, China, pp. 67-68.
- Zhou X, Yuan Y, Yang Y, Rutzke M, Thannhauser TW, Kochian LV, Li L. 2009. Involvement of a Broccoli COQ5 Methyltransferase in the Production of Volatile Selenium Compounds. Plant Physiol 151: 528-540.
- Sooksanguan T, Yakubov B, Kozlovskyy VI, Barkume CM, Howe KJ, Thannhauser TW, Rutzke MA, Hart JJ, Kochian LV, Rea PA, and OK Vatamaniuk. 2009. Drosophila ABC Transporter, DmHMT-1, Confers Tolerance to Cadmium. DmHMT-1 and its yeast homolog, SpHMT-1, are not essential for vacuolar phytochelatin sequestration. J. Biol Chem 284: 354-362.
- Kochian LV, Hoekenga OA, Magalhaes JV, Pineros MA. 2009. Maize aluminum tolerance. In: The Maize Handbook - Volume 1: The Biology of Maize (Bennetsen J, and Hake, S,eds). Springer-Verlag, New York, NY, pp. 367-380.
- Liu JW, Magalhaes JV, Shaff JE, Kochian LV. 2009. Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance. Plant Journal 57: 389-399.
- Ligaba A, Kochian LV, Pineros MA. 2009. Phosphorylation at S384 regulates the activity of the TaALMT1 malate transporter that underlies aluminum resistance in wheat. Plant Journal 60: 411-423.
- Wang Y-H, Mosebach CM, Kibbey AS, Ryhal MK, Jones AD, Kochian LV. 2009. Generation of Arabidopsis mutants by heterologous expression of a full length cDNA library from tomato fruits. Plant Mol Biol Reporter 27: 454-461.
- Kuepper H, and LV Kochian. 2010. Transcriptional regulation of metal transport genes and mineral nutrition during acclimation to cadmium and zinc in the Cd/Zn hyperaccumulator, Thlaspi caerulescens (Ganges population). New Phytologist 185: 114-129.
- Liu JW, Magalhaes JV, Shaff JE, Kochian LV. 2009. Arabidopsis combines the function of aluminum-activated citrate and malate transporters from the MATE and ALMT families in aluminum tolerance. Proceedings of the 7th International Symposium on Plant-Soil Interactions at Low pH, Guangzhou, China, pp. 69-70.
- Kochian LV, Magalhaes JV, Liu J, Schaffert RE, Guimaraes CT, Alves VMC, Klein PE. The sorghum aluminum tolerance gene, SbMATE. 2009. US Patent # US 7,582, 809 B2. Awarded 9-1-09
- Kochian LV, Magalhaes JV, Liu J, Hoekenga OA, Pineros MA. 2009. Recent advances on the molecular basis of crop aluminum resistance. Proceedings of the 7th International Symposium on Plant-Soil Interactions at Low pH, Guangzhou, China, pp. 1-2.
- Hoekenga OA, Buckler ES, Kirst M, Krill AM, Lyi SM, Magalhaes JV, Maron LG, Kochian LV. 2009. Joint linkage-association analysis of aluminium tolerance in maize. Proceedings of the 7th International Symposium on Plant-Soil Interactions at Low pH, Guangzhou, China, pp. 136-137.
- Pineros MA, Kochian LV. 2009. Overview of the structure-function relations underlying the functionality of ALMT and MATE-type transporters involved in the organic acid release Al tolerance response. Proceedings of the 7th International Symposium on Plant-Soil Interactions at Low pH, Guangzhou, China, pp. 55-56.
- Ligaba A, Pineros MA, Kochian LV. 2009. Modulation of TaALMT1 transport activity by protein phosphorylation. . Proceedings of the 7th International Symposium on Plant-Soil Interactions at Low pH, Guangzhou, China, pp. 73-74.
- Shaff JE, Schultz BA, Craft EJ, Clark RT, Kochian LV. 2009.GEOCHEM-EZ: A chemical speciation program with greater power and flexibility. Proceedings of the 7th International Symposium on Plant-Soil Interactions at Low pH, Guangzhou, China, pp. 27-28.
Progress 10/01/07 to 09/30/08
OUTPUTS: For the sorghum aluminum (Al) tolerance research, we continued to characterize the major sorghum Al tolerance gene we cloned last year, SbMATE, which encodes a MATE transporter that mediates 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 SbMATE, where each NIL harbors a different version of SbMATE generated from backcrossing different sorghum lines into the same recurrent parent. Using these lines, we are identifying the most effective versions of this gene for enhancing Al tolerance to be used in molecular breeding programs. Al tolerance in maize is a complex, quantitative trait involving multiple genes, unlike sorghum where a single tolerance gene is usually involved in any specific line. 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. We have also conducted a detailed analysis of root gene expression under Al stress using maize gene chips with the Al-tolerant and sensitive parents of the mapping population. To facilitate large scale screening of plants for Al tolerance and root growth and architecture in general, we have developed new tools to phenotype root systems. We have developed custom 2D imaging and software tools to capture and analyze whole root systems. Working with hydroponic growth systems we designed a flexible growth setup that allows plants to be grown and photographed with minimal disturbance to their root systems. To capture high quality root images, we constructed a custom imaging system that takes advantage of improved optics and unique lighting techniques to provide high contrast images of both coarse (thicker roots) and fine root systems. Using the NetBeans 6.0 IDE and standard Java libraries, we developed RootReader2D software to assist in quantifying individual and whole root system lengths. This software provides an interactive platform in which root lengths can be measured and recorded from root images. While the ability to measure whole root systems as well as specific roots of interest was the main purpose for creating this software, other functions such as lateral root counters, width measuring and mathematical descriptors are being added. Additionally, the ability to batch process images to acquire total root system lengths has been integrated and used. Building off of prior silhouette-based 3D root imaging work (Fang, 2006), optical and mechanical systems with improved reliability were developed. Proof of concept work was performed using an optical correction tank and improved axis-of-rotation calibration techniques. Using maize and rice root systems grown in enclosed glass cylinders containing nutrient enriched gellan gum media, 3D reconstructions of growing root systems were generated. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
From our research on sorghum Al tolerance, we have obtained clear evidence for the existence of several new Al tolerance genes in sorghum that contribute to the significant variation in Al tolerance across a large number of different sorghum lines. Furthermore, we have found strong evidence that these novel tolerance genes interact with our previously identified Al tolerance gene SbMATE to facilitate maximal SbMATE gene expression and tolerance. It was found that several maize members of the MATE gene family, for which our major sorghum Al tolerance gene, SbMATE, is also a member, 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 gene we have designated ZmMATE1. We also identified a second related MATE, ZmMATE2, that is also differentially expressed in root tips of tolerant lines. Genetic mapping of ZmMATE1 confirmed it is located at the major Al tolerance QTL on chromosome 6, while ZmMATE2 maps to the same location as the second major Al tolerance QTL on chromosome 5. Hence these are strong candidates for the first identified maize Al tolerance genes. For our work on root system imaging and phenotyping, 2D imaging and analysis tools to assist in high throughput phenotyping were created and tested using both coarse (thicker roots) and fine root systems. Minimal post-processing of the root images was required before software analysis. To facilitate individual root measurements, RootReader2D software was designed to automatically predict root path and length from user provided start and end points. The ability to efficiently modify the generated path was incorporated to correct any erroneous predictions. Batch image processing capabilities were added to provide automated calculation of total root system lengths. System validation and calibration was performed using a calibration grid, wires and roots with known lengths. When comparing RootReader2D length measurements to traced measurements using ImageJ software, no significant differences were observed. Improvements to the 3D imaging and calibration system resulted in increased quality and reliability of our 3D root reconstructions. Correcting for optical distortion proved essential when using light-based techniques with cylindrical growth systems. Axis-of-rotation calibration techniques provide a simple and efficient way to calibrate our 3D systems. Using improved systems, we successful captured and reconstructed both fine and large root systems growing over several days. When using properly corrected and calibrated systems, the 3D reconstruction of growing root systems will provide great opportunities to study the dynamic growth responses of roots to acid soils as well as other stress environments.
- Parameswaran A, Leitenmaier B, Yang M, Welte W, Kroneck PMH, Papoyan A, Kochian LV, Kupper H. 2007. A native Zn/Cd transporting P1B ATPase from natural overexpression in a hyperaccumulator plant reveals post-translational processing. Biochem Biophys Research Comm 363: 51-56.
- Pineros MA, Cancado GMA, Maron LG, Lyi MS, Menossi M, Kochian LV. 2008. Not all ALMT1-type transporters mediate aluminum-activated organic acid responses: The case of ZmALMT1. Plant Journal 53: 352-367.
- Klein MA, Sekimoto H, Milner MJ and LV Kochian. 2008. Investigation of heavy metal hyperaccumulation at the cellular level: development and characterization of Thlaspi caerulescens suspension cell lines. Plant Physiol 147: 2006-2016.
- Maron LG, Kirst M, Mao C, Milner MJ, Menossi M and LV. Kochian. 2008. Transcriptional profiling of aluminum toxicity and tolerance responses in maize roots. New Phytologist 179: 116-128.
- Pineros MA, Kochian LV. 2008. Novel properties of the wheat aluminum tolerance organic acid transporter (TaALMT1) revealed by electrophysiological characterization in Xenopus oocytes: Functional and structural implications. Plant Physiol 147: 2131-2146.
Progress 10/01/06 to 09/30/07
We have completed the verification that the candidate sorghum aluminum (Al) tolerance gene, AltSB, which we isolated via positional cloning in sorghum, was a major sorghum Al tolerance gene. The verification involved analysis of homozygous transgenic T3 Arabidopsis lines expressing AltSB, which exhibited large increases in Al tolerance. These same lines also showed a large increase in Al-activated root citrate exudation, verifying that a member of the Multidrug and Toxic Compound (MATE) family is a citric acid efflux transporter that confers Al tolerance in sorghum .The verification also involved AltSB expression in wheat. Transgenic T1 lines of Bobwhite wheat expressing AltSB were significantly more Al tolerant than wild type Bobwhite wheat lines, showing the possible utility of using this gene for improving cereal Al tolerance in general. We also completed an initial characterization of the regulation/function of AltSB, which showed that differences in AltSB
expression underlie the significant allelic variation for this gene which is responsible for much of the variation in sorghum Al tolerance. Preliminary diversity analysis pointed to polymorphisms in two regulatory regions, one in the promoter (a MITE containing region) and polymorphisms in the 2nd intron, as important for differences in AltSB expression associated with differential Al tolerance. This work was published in Nature Genetics (39: 1156-1161) and featured in the "Leading Edge" section of the journal, Cell (130: 965). In Caniato et al. (TAG 114: 863; 2007) we reported on a comprehensive study on genetic diversity for Al tolerance in sorghum, indicating there is both significant allelic and non-allelic heterogeneity for Al tolerance in sorghum, which can be used for breeding highly Al tolerant sorghums. Therein we showed the Al tolerance results for 3 near-isogenic lines in which the AltSB allele from different Al tolerant sources were introgressed into the same Al sensitive
genetic background. We have now expanded our collection to a total of 9 NILs including those carrying the elite alleles from SC566 and SC283. We also identified a homolog of AltSB in Arabidopsis, AtMATE, that encodes a root Al-activated citrate transporter involved in Arabidopsis Al tolerance. Together with our previously characterized Arabidopsis Al tolerance gene that is an Al-activated malate transporter encoded by AtALMT1, this discovery has allowed us allows to examine the relations and interactions between the Al-activated citrate and malate transport systems in the same plant species. When we knock out the AtMATE via T-DNA insertion into its promoter, this abolishes the Al-inducibility of the AtMATE gene expression as well as the Al-activated citrate exudation under Al stress. The AtALMT1/AtMATE double mutant is deprived of both Al-activated malate and citrate exudation from roots, and this mutant is extremely hypersensitive to Al stress. Thse findings indicate that the
AtALMT1-mediated Al-activated malate exudation and the AtMATE-mediated Al-activated citrate exudation are independent traits, which is consistent with the non-orthologous nature of these two gene families.
The cloning of the novel sorghum aluminum tolerance gene opens up new avenues of research for improving crop production on acid soils in developing copuntries. A patent applicatioin has been field for this purpose, and we are now working with sorghum breeders in Africa to identify easy to use molecular markers for the best alleles of our sorghum Al tolerance gene to quickly facilitate improved Al tolerance for sorghum grown in Africa. From the analysis of Al tolerance in the above-descrobed NIL set and respective parents we will be able to select elite AltSB alleles as well as new sources of Al tolerance carrying different Al tolerance genes. This information will be used for a reasonable choice of recurrent parents in backcross breeding programs as well as new Al tolerance sources for gene pyramiding schemes. The discovery of the sorghum Al tolerance gene has opened up new avenues of research aimed at identifying candidate Al tolerance genes in other plant species that
are MATE homologs. In maize, we have identified an AltSB homolog that is a strong candidate for a maize Al tolerance gene that mediates Al-activated root citrate efflux in maize. The gene is preferentially expressed in the root tips of Al tolerant maize lines, and is strongly Al induced only in tolerant lines. Mapping of this maize MATE gene to Embrapa's set of Al tolerance related RILs has shown that the gene maps directly within a major Al tolerance QTL on maize chr 6
- Lyi SM, Zhou X,. Kochian LV, Li L. 2007. Biochemical and molecular characterization of the homocysteine S-methyltransferase from broccoli (Brassica oleracea var. italica. Phytochem 68: 1112-1119.
- Papoyan A, Pineros MA, Kochian LV. 2007. Plant Cd2+ and Zn2+ status effects on root and shoot heavy metal accumulation in Thlaspi caerulescens. New Phytol 175: 51-58.
- Magalhaes JM, Liu J, Guimares CT, Lana UGP, Alves VM, Wang Y-H, Schaffert RE, Hoekenga OA, Shaff JE, Pineros MA, Klein PE, and LV Kochian. 2007. A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum.. Nature Genetics 39: 1156-1161.
- Kobayasi Y, Hoekenga OA, Ito H, Nakashima M, Saito S, Shaff JE , Maron LG, Pineros MA, Kochian LV, Koyama H. 2007. Characterization of AtALMT1 expression in aluminum inducible malate release and its role for rhizotoxic stress tolerance in Arabidopsis thaliana. Plant Physiol 145: 843-852.
- Caniato FF, Guimaraes CT, Schaffert RE, Alves VMC, Kochian LV, Borem A, Klein PE, Magalhaes JV. 2007. Genetic diversity for aluminum tolerance in sorghum. Theor Applied Genetics 114: 863-867.
- Kupper H, Seib LO, Sivaguru M, Hoekenga OA, and LV Kochian. 2007. A novel method for quantitative in situ hybridization in plants reveals regulation of a zinc transporter in the Cd/Zn hyperaccumulator Thlaspi caerulescens (Ganges). The Plant Journal 50: 159-175.
Progress 01/01/06 to 12/31/06
Considerable progress was made in the areas of sorghum aluminum (Al) tolerance. With regards to sorghum Al tolerance research, in the last year, we cloned a major aluminum (Al) tolerance gene from sorghum, AltSB via high resolution mapping of a 2300 F2 population generated from a cross between SC283, a standard line for sorghum Al tolerance, and BR007, which is a very Al sensitive sorghum line. The gene is a member of the MATE (Multidrug and Toxin Efflux) family of membrane transporters and appears to encode a novel citrate efflux transporter that is activated by Al (we also have shown that root Al-activated citrate efflux is the mechanism of sorghum Al tolerance). In 2006, we verified that this gene underlies the AltSB locus, and have used further high resolution mapping and allelic variation at the locus to elaborate a list of the sequence polymorphisms that could be associated with the Al tolerance phenotype.To verify that AltSB encodes a plant Al tolerance gene,
we expressed AltSB in Arabidopsis behind the cauliflower mosaic virus CaMV 35S promoter and have scored homozygous T3 lines for Al tolerance, in order to quickly assess the functionality of AltSB. We have obtained a number of homozygous T3 lines in Arabidopsis where expression of AltSB has conferred a significant increase in Al tolerance. We also found that expression of AltSB conferred Al-activated root citrate efflux, which is the sorghum Al tolerance mechanism. These findings strongly indicate that AltSB encodes an Al-activated root citrate efflux transporter and is the major sorghum Al tolerance gene. We also verified that AltSB is the sorghum Al tolerance gene by quantifying its expression in root tips (the site of Al tolerance and toxicity) of tolerant versus sensitive near isogenic lines (NILs) of sorghum. Quantitative real-time PCR analysis of candidate gene expression indicates that AltSB is expressed strongly in the root tip of the Al tolerant NIL, while its expression is
not detectable in the root tip of the sensitive NIL. Furthermore, expression of the MATE is Al-inducible, and the degree of inducibility increases strongly with longer periods of root exposure to Al. This correlates nicely with our recent findings regarding the physiology of sorghum Al tolerance, in that sorghum Al tolerance is induced by Al in the tolerant genotypes. After 1 day in Al, root growth is inhibited by approx. 55 percent, while by day three, Al tolerance increases moderately (40 percent inhibition). By 6 days in Al there is a strong increase in tolerance, as no inhibition of root growth by Al is observed. Also, we have determined that the likely mechanism of Al tolerance is Al-activated root citrate exudation, and this citrate exudation is also induced by Al. That is, the rate of Al-activated citrate release is low after 1 day in Al, and increases 4- and 10 fold, respectively, after 3 and 6 days of Al exposure (Magalhaes et al, in preparation). These findings further
verify that our candidate MATE gene for the AltSB locus encodes a root citrate efflux transporter that is Al-inducible at the level of gene transcription, and is also Al-activated at the level of protein function (transport).
The broader significance of this work resides in the fact that aluminum toxicity occurs in acid soils which are quite extensive in the world, comprising as much as 50 percent of the world's potentially arable lands, mostly in the tropics and subtropics. Novel, genomics-based strategies to cope with Al stress are now possible, which will help to increase crop yields on Al-toxic acid soils which are a major limiting factor for agriculture in developing countries where food security is most tenuous. It has been estimated that aluminum toxicity as a worldwide agronomic problem is only exceeded by drought stress with regards to abiotic limitations to crop production. Because the gene described in this annual report is a novel Al tolerance gene, its discovery and characterization represent a major advance for a field of research that has large implications for agriculture worldwide, and particularly in developing countries.
- Liao H, Wan H, Shaff JE, Wang X, Yan X, and Kochian LV. 2006. Phosphorus and aluminum ınteractions in soybean in relation to Al tolerance: Exudation of specific organic acids from different regions of the ıntact root system. Plant Physiol 141: 674-684.
- Hoekenga OA, Maron LG, Pineros MA, Cancado GMA, Shaff JE, Kobayashi Y, Ryan PR, Dong B, Delhaize E, Sasaki T, Matsumoto H, Koyama H, and LV Kochian. 2006. AtALMT1 (At1g08430) is a novel, essential factor for aluminum tolerance in Arabidopsis thaliana and encodes an aluminum-activated malate transporter. Proc Natl Acad Sci USA 103: 9738-9743.
Progress 01/01/05 to 12/31/05
We have made significant progress on sorghum and Arabidopsis aluminum (Al) tolerance, and mechanisms of heavy metal hyperaccumulation in the Zn/Cd hyperaccumulating plant species, Thlaspi caerulescens. In sorghum, the first bona fide plant Al tolerance gene was cloned in sorghum via map-based cloning approaches. This gene conditions the major sorghum Al tolerance locus, AltSB. AltSB encodes a novel membrane transporter and we have shown that is the transporter that mediates Al-activated citrate efflux from the sorghum root tip, which we have also shown to be the physiological mechanism of sorghum Al tolerance. The major difference in AltSB in tolerant versus sensitive genotypes is in expression and not function, in that it is expressed primarily in the root tip of only the Al tolerant sorghum genotypes. We have identified a MITE (miniature inverted repeat-type transposable element) in the promoter of this gene that is associated with the difference in expression, and
may be the enhancer element that drives high expression in tolerant sorghum genotypes. In Arabidopsis, we showed that a homolog of a putative wheat Al tolerance gene, ALMT1 (for Aluminum-activated Malate Transporter) plays the same role in Arabidopsis and is critical for Arabidopsis Al tolerance. This ALMT1 homolog maps close to two major Al tolerance QTL we identified on chromosome 1, but is not the gene responsible for either QTL. We are in the process of identifying the genes responsible for these two QTL. It appears that there is an Al tolerance box on chromosome 1 that contains at least 3 genes all located near each other and involved in Al tolerance. A novel heavy metal ATPase, TcHMA4 (for Thlaspi caerulescens Heavy Metal ATPase) that can transport Cd, Pb, and Zn out of plant cells, was isolated and characterized . We showed that this transporter is the xylem pump involved in transporting heavy metals and micronutrients from roots to the leaves in Thlaspi and plays a key role in
the heavy metal hyperaccumulation phenotype in Thlaspi. We also identified regions of the 5' cytoplasmic tail of the TcHMA4 protein and we showed that these regions function as heavy metal binding domains. In yeast, expression of these peptides derived from the entire TcHMA4 protein conferred a very high degree of heavy metal tolerance and accumulation in yeast. We have now overexpressed these peptides in Arabidopsis and currently are testing whether they can significantly enhance plant heavy metal tolerance and metal hyperaccumulation.
This discovery of this plant Al tolerance gene opens up research aimed at elucidating the molecular mechanism of Al tolerance in plants, as well as for providing a tool for improving the acid soil tolerance of crop plants both via biotechnology and marker-assisted selection. The work on the HMA4 gene in Thlaspi has identified peptides that might be useful, via biotechnology, to enhance the metal detoxictying and accumulating properties of plants. If so, these peptides may be useful via biotechnology in improving the phytoremediation potential of plants. An invention disclosure report on this finding has been submitted to USDA-ARS (the agency Dr. Kochian works for) as the first step towards obtaining a patent.
- Hart JJ, Welch RM, Norvell WA, Clarke JM, and LV Kochian. 2005. Zinc effects on cadmium accumulation and partitioning in near isogenic lines of durum wheat that differ in grain cadmium concentration. New Phytologist 167: 391-401
- Kochian LV, Pineros MA, Hoekenga OA. 2005. The physiology, genetics and molecular biology of plant aluminum resistance and toxicity. Plant and Soil 274: 175-195.
- Raman H, K Zhang, M Cakir, R Appels, JS Moroni, LG Maron, LV Kochian, R Raman, I Muhammad, F-D Brockman, DF Garvin, I Waters, P Martin, T Sasaki, Y Yamamoto, H Matsumoto, DM Hebb, E Delhaize, and PR Ryan. 2005. Molecular characterization and mapping of ALMT1, the aluminium-tolerance gene of bread wheat (Triticum aestivum L.). Genome 48: 781-791.
- Kochian L, Hoekenga OA, Magalhaes J, Pineros M, Alves V, Maron L, Mason P, Guimares C, and Schaffert R. 2005. Integrating genomics, molecular genetic and physiological approaches to identify plant aluminum tolerance genes and their associated physiological mechanisms. In: Plant Nutrition for Food Security, Human Health, and Environmental Protection. CJ Li, FS Zhang et al., eds., Tsinghua University Press, Beijing, China. Pp. 18-20.
Progress 01/01/04 to 12/31/04
Progress was made on crop aluminum (Al) tolerance including: 1) Map-based cloning of the sorghum Al tolerance gene, AltSB. In 2004 we sequenced the BAC harboring AltSB which enabled us to identify 17 putative genes on the BAC. Each of these ORFs is being cloned from the Al tolerant and sensitive parents of our mapping population, and this has enabled us to identify several new markers on the BAC. Based on fine scale mapping with these markers we have identified flanking markers on the BAC that flank a 70 kb interval that contains 7 genes, one of which must be AltSB. Based on annotation, we have identified a strong candidate for AltSB, a gene encoding a MATE or Multidrug and Toxin Efflux transporter. Subsequent sequence and expression analysis have strengthened the case for this transporter being the sorghum Al tolerance gene, and this is currently being verified via expression in transgenic sorghum, maize, and Arabidopsis. 2) In Arabidopsis, we showed that a homolog
of a putative wheat Al tolerance gene, ALMT1(for Aluminum-activated Malate Transporter) plays the same role in Arabidopsis and is critical for Arabidopsis Al tolerance. This ALMT1 homolog maps close to two major Al tolerance QTL we identified on chromosome 1, but is not the gene responsible for either QTL. We are in the process of identifying the genes responsible for these two QTL. It appears that there is an Al tolerance box on chromosome 1 that contains at least 3 genes all located near each other and involved in Al tolerance. Progress was also made on heavy metal/micronutrient hyperaccumulation in Thlaspi caerulescens including: 1) Identification of a novel heavy metal ATPase, TcHMA4 (for Thlaspi caerulescens Heavy Metal ATPase) that can transport Cd, Pb, and Zn out of plant cells. We showed that this transporter is the xylem pump involved in transporting heavy metals and micronutrients from roots to the leaves in Thlaspi and plays a key role in the heavy metal hyperaccumulation
phenotype in Thlaspi. 2) Identification of regions of the 5' cytoplasmic tail of the TcHMA4 protein and we showed that this region function as a heavy metal binding domain. In yeast, expression of these regions of the overall tcHMA4 protein as individual peptides conferred a very high degree of heavy metal tolerance and accumulation in yeast. We have now overexpressed these peptides in Arabidopsis and currently are testing whether they can significantly enhance plant heavy metal tolerance and metal hyperaccumulation. If so, they may be useful via biotechnology in improving the phytoremediation potential of plants. An invention disclosure report is being filed on this finding as the first step towards submitting a patent application.
The research on Al tolerance is moving us closer to our goal of isolating important Al tolerance genes. These genes should be powerful tools for improving Al tolerance in a number of economically important crop species, and thus improve agriculture on the acid soils that comprise up to 50% of the arable lands in the world. The moelcular research on heavy metal hyperaccumulating plant species is identifying genes that confer the ability to mie toxic metals from the soil and deposit them in the shoot biomass, which is easily harvestable for storage in a toxic metal repository. These genes will comprise a molecular tool box for use in developing transgenic plants well suited for the phytoremediation of toxic metal contaminated soils.
- Magalhaes, J.V., Garvin, D.F., Sorrells, M.E., Klein, P.E., Schaffert, R.E., Wang, Y.-H., Li, L., and Kochian, L.V. 2004. Comparative mapping of a major aluminum tolerance gene in sorghum and other species in the Poaceae. Genetics 167:1905-1914.
- Kochian, L.V., Hoekenga, O.A., and Pineros, M.A. 2004. How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorous efficiency. Ann. Rev. Plant Biol. 55:459-493
- Hacisalihoglu, G., Hart, J.J., Vallejos, E., and Kochian, L.V. 2004. The role of shoot-localized processes in the mechanism of Zn efficiency in common bean. Planta 218:704-711
- Cohen, C.K., Garvin, D.F., and Kochian, L.V. 2004. Kinetic properties of a micronutrient transporter from Pisum sativum indicate a primary function in Fe uptake from the soil. Planta 218:784-792.
- Huang, J.H., Poynton, C.Y., Kochian, L.V., and Elless, M.P. 2004. Phytofiltration of arsenic from drinking water using an arsenic-hyperaccumulating fern. J Env Qual 38:3412-3417.
- Poynton, C.Y., Huang, J.W., Blaylock, M.J., Kochian, L.V., and Elless, M.P. 2004. Mechanisms of arsenic hyperaccumulation in Pteris species: Root As influx and translocation. Planta 219: 1080-1088.
- Papoyan, A., and Kochian, L.V. 2004. Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance: Characterization of a novel heavy metal transporting ATPase. Plant Physiol 136:3814-3823.
- Pineros, M.A., Shaff, J.E., Manslank, H.S., Alves, V.M.C., and Kochian, L.V. 2004. Aluminum resistance in maize can not be solely explained by root organic acid exudation: A comparative physiological study. Plant Physiol 137:231-241.
Progress 01/01/03 to 12/31/03
The goals of this project are 1) to identify the suite of genes conferring Zn hyperaccumulation in the metal hyperaccumulating plant species, Thlaspi caerulescens, in order to develop strategies both to improve Zn content in plant-based foods and to develop plants better suited for the remediation of surface soils contaminated with heavy metals; and 2) to elucidate the mechanisms and the underlying genes crop plants employ to tolerate toxic levels of aluminum (Al) on acid soils. In 2003, the research on micronutrient and heavy metal hyperaccumulation focused on the molecular basis for the extreme heavy metal/micronutrient accumulation in the shoots of Thlaspi caerulescens. We characterized a novel heavy metal-transporting ATPase that appears to be the key transporter that efficiently moves that heavy metals absorbed from the soil from the root to the shoot. The research on Al tolerance focuses on integrating molecular and genetic studies of Al tolerance in populations
of sorghum, maize, and Arabidopsis segregating for Al tolerance with physiological studies identifying mechanisms of Al tolerance. We are also using several different molecular approaches aimed at cloning Al tolerance genes, which ultimately will be used to improve this trait in more Al sensitive cereal crops and other food crops. In a project aimed at cloning the major Al tolerance gene in sorghum, we have mapped the Al tolerance locus to a single sorghum BAC and are sequencing this BAC. In Arabidopsis we have identified a candidate Al tolerance gene that appears to be the Al-activated malate transporter that is critical to the tolerance mechanism in Arabidopsis.
The research on Al tolerance is moving us closer to our goal of isolating important Al tolerance genes. These genes should be powerful tools for improving Al tolerance in a number of economically important crop species, and thus improve agriculture on the acid soils that comprise up to 50% of the arable lands in the world.
- Hacisalihoglu, G., Hart, J.J., Wang, Y.-H., Cakmak, I., and Kochian, L.V. 2003. Zinc efficiency is correlated with enhanced expression and activity of Zn-requiring enzymes in wheat. Plant Physiology 131: 595-602.
- Pineros, M.A. and Kochian, L.V. 2003. Differences in whole cell and single channel ion currents across the plasma membrane of mesophyll cells from two closely related Thlaspi species. Plant Physiology 131: 583-594.
- Wang, Y.-H., Kochian, L.V., Doyle, J.F., and Garvin, D.F. 2003. Differential regulation of two tomato nonsymbiotic hemoglobin genes in response to diverse changes in nutrient status. Plant Cell Environ 26: 673-680.
- Levent, O., Cakmak, I., and Kochian, L.V. 2003. Shoot biomass and zinc/cadmium uptake for hyperaccumulator and non-accumulator Thlaspi species in response to growth on a zinc-deficient calcareous soil. Plant Science 164: 1065-1071.
- Hoekenga, O.A., Vision, T.J., Shaff, J.E., Monforte, A.J., Lee, G.P., Howell. S.H., and Kochian, L.V. 2003. Identification and characterization of Al tolerance loci in Arabidopsis thaliana (Landsberg x Columbia) by quantitative trait locus mapping: A physiologically simple but genetically complex trait. Plant Physiology 132: 936-948.
- Hacisalihoglu, G., and Kochian, L.V. 2003. How do some plants tolerate low levels of soil zinc? Mechanisms of zinc efficiency in crop plants. New Phytologist 159: 341-350.
- Fuhrmann, M., Lasat, M., Ebbs, S., Cornish, J., and Kochian, L.V. 2003. Uptake and release of cesium-137 by five plant species as influenced by soil amendments in field experiments. J Environ Qual 32: 2272-2279.
Progress 01/01/02 to 12/31/02
The goals of this project are 1) to identify the suite of genes conferring Zn hyperaccumulation in the metal hyperaccumulating plant species, Thlaspi caerulescens, in order to develop strategies both to improve Zn content in plant-based foods and to develop plants better suited for the remediation of surface soils contaminated with heavy metals; and 2) to elucidate the mechanisms and the underlying genes crop plants employ to tolerate toxic levels of aluminum (Al) on acid soils. In 2002, the research on micronutrient and heavy metal hyperaccumulation focused on the molecular basis for the extreme heavy metal/micronutrient accumulation in the shoots of Thlaspi caerulescens. We cloned several genes novel that appear to play a role in the extreme metal tolerance exhibited by Thlaspi, including a heavy metal-transporting ATPase and a number of metallothionein genes. We currently are characterizing the functions of the proteins encoded by these genes. The research on Al
tolerance focuses on integrating molecular and genetic studies of Al tolerance in populations of sorghum, maize, and Arabidopsis segregating for Al tolerance with physiological studies identifying mechanisms of Al tolerance. We are also using several different molecular approaches aimed at cloning Al tolerance genes, which ultimately will be used to improve this trait in more Al sensitive cereal crops and other food crops. In a project aimed at cloning the major Al tolerance gene in sorghum, we have identified several molecular markers closely linked to the Al tolerance locus and are proceeding with fine scale mapping to ultimately clone this gene. In Arabidopsis we have identified two regions of the Arabidopsis genome on chromosomes 1 and 5 that harbor major Al tolerance genes. We have shown that both genes act together in a mechanism involving Al-activated exudation of malate from the root tip. The excreted malate binds Al outside the root and detoxifies it. We are currently working
on cloning these Al tolerance genes.
The research on Al tolerance is moving us closer to our goal of isolating important Al tolerance genes. These genes should be powerful tools for improving Al tolerance in a number of economically important crop species, and thus improve agriculture on the acid soils that comprise up to 50% of the world's arable lands.
- Ebbs, S.D., Lau I., Ahner B., and Kochian, L.V. 2002. Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator, Thlaspi caerulescens. Planta 214: 635-640.
- Pineros M.A., Magalhaes J.V., Alves V.M., and Kochian L.V. 2002. A physiological and biophysical examination of aluminum tolerance in maize suggests the existence of multiple tolerance mechanisms. Plant Physiol 129: 1194-120.
- Tang Y., Carver, B.F., Sorrells, M.E., Kochian, L.V., and Garvin, D.F. 2002. Physiological genetics of mechanisms associated with aluminum tolerance in the wheat cultivar Atlas 66. Crop Sci 42: 1541-1546.
- Wang, Y.-H., Garvin D.F., and Kochian L.V. 2002. Rapid induction of regulatory and transporter genes in response to phosphorous, potassium and iron deficiencies in tomato roots. Evidence for cross talk and root/rhizosphere mediated signals. Plant Physiol 130: 1361-1370.
Progress 01/01/01 to 12/31/01
The goals of this project are 1) to identify the suite of genes conferring Zn hyperaccumulation in the metal hyperaccumulating plant species, Thlaspi caerulescens, in order to develop strategies both to improve Zn content in plant-based foods and to develop plants better suited for the remediation of surface soils contaminated with heavy metals; and 2) to elucidate the mechanisms and the underlying genes crop plants employ to tolerate toxic levels of aluminum (Al) on acid soils. In 2001, the research on micronutrient and heavy metal hyperaccumulation focused on the molecular basis for the extreme heavy metal/micronutrient accumulation in the shoots of Thlaspi caerulescens. We previously found that altered regulation of key micronutrient transport genes plays a role in this trait. Thus, in 2001 we cloned a transcription factor that appears to regulate micronutrient transporter gene expression via changes in plant micronutrient status. It appears that this gene is an
important molecular regulator of micronutrient homeostasis, and alterations in this protein appear to confer heavy metal hyperaccumulation and tolerance. This gene may prove to be a useful molecular tool for improving plants for the phytoremediation of heavy metal contaminated soils. The research on Al tolerance focuses on mapping of Al tolerance genes in populations of wheat and other cereals segregating for Al tolerance with identification and characterization of physiological mechanisms of Al tolerance. We are also using several different molecular approaches aimed at cloning Al tolerance genes, which ultimately will be used to improve this trait in more Al sensitive cereal crops and other food crops. In 2001, we made significant progress on research aimed at ultimately cloning Al tolerance genes. In a project aimed at cloning the major Al tolerance gene in sorghum, we have identified several molecular markers closely linked to the Al tolerance locus. In collaboration with Dr. John
Mullet of Texas A&M University we have determined the physical location of these markers in the sorghum genome and have identified additional closely linked markers, in order to conduct fine-scale mapping and map-based cloning of the sorghum Al tolerance gene. In the research project focusing on the molecular basis for the extreme heavy metal/micronutrient accumulation in the shoots of Thlaspi caerulescens, we previously found that altered regulation of key micronutrient transport genes plays a role in this trait. Other research on Al tolerance involved a major mechanism of aluminum (Al) tolerance in crop plants based on Al activated exudation of Al binding organic acids from the root apex. We isolated genes encoding anion channels that could facilitate this organic acid release from the root tip of a very Al tolerant wheat genotype. We now have isolated 8 different anion channel genes and are in the process of characterizing these genes to determine if any of them encode the
Al-activated organic acid transporter that mediates Al-induced malic acid release from the root tip of Al tolerant wheat. These studies may enable us to identify key molecular determinants of Al tolerance in cereal crops.
: The research on Al tolerance is moving us closer to our goal of isolating important Al tolerance genes. These genes should be powerful tools for improving Al tolerance in a number of economically important crop species, and thus improve agriculture on the acid soils that comprise up to 50% of the world's arable lands.
- Hacisalihoglu G. Hart JJ, Kochian LV. 2001. High and low affinity zinc transport systems and their possible role in zinc efficiency in bread wheat (Triticum aestivum L.). Plant Physiol 125: 1-8.
- Wang, Y.-H., Garvin D.F., and Kochian L.V. 2001. Nitrate-induced genes in tomato roots: Array analysis reveals novel genes that may play a role in nitrogen nutrition. Plant Physiology 127: 345-359.
- Lasat MM, Pence NS, Letham DLD, and Kochian LV. 2001. Zinc phytoextraction in Thlaspi caerulescens. Int J Phytoremediation 3: 129-144.
- Pineros MA, Kochian LV. 2001. A patch clamp study on the physiology of aluminum toxicity and tolerance in Zea mays: Identification and characterization of Al3+-induced anion channels. Plant Physiol 124: 1-14.
- Papernik LA, Bethea AS, Singleton TE, Magalhaes JV, Garvin DF and Kochian LV. 2001. Physiological basis of reduced Al tolerance in ditelosomic lines of Chinese Spring wheat. Planta 212: 829-834
Progress 01/01/00 to 12/31/00
Research on this project has focused on: 1)identifying mechanisms and genes controlling plant heavy metal tolerance and hyperaccumulation both to use in developing plants better suited for remediating contaminated soils and to reduce the entry of heavy metals into the food chain; 2) identifying mechanisms and genes regulating of beta-carotene accumulation to use in improving beta-carotene content in plant foods; and 3) identifying mechanisms and genes controlling tolerance to aluminum toxicity to use in developing crops better suited for growth on acid soils that make up 50% of world's potentially arable lands. Significant accomplishments include: 1) The cloning and characterization of genes encoding heavy metal/micronutrient transporters from the heavy metal hyperaccumuating plant, Thlaspi caerulescens, showed that regulation of genes involved in micronutrient nutrition was altered in this plant, causing the upregulation of a number of micronutrient transporters and
genes involved in metal tolerance. This finding has led us to look for key regulatory gene(s) whose alteration may provide us with the molecular switch needed to transform normal plants into micronutrient and heavy metal hyperaccumulators. 2) research into the Or mutant, a single gene mutation in cauliflower that causes massize beta-carotene accumulation in plant tissues that normally do not accumulate this vitamin A precursor demonstrated that the beta-carotene accumulates in large crystalline sheets within plastids, and that the Or gene appears to induce the formation of these sheets by directing plastids to become chromoplasts. This is significant because it suggests that if the Or gene can be cloned, it may direct this same phenomenon within plastids of the endosperm of grains such as wheat and rice;3) Research on the genetic and physiological complexity of Al tolerance in wheat showed that Al tolerance in wheat involves the functioning of several genes that all act in concert to
enhance a single Al tolerance mechanism based on Al-induced release of Al-detoxifying malic acid from the root tip. These findings have led us to focus on this particular tolerance mechanism for our efforts to clone Al tolerance genes in wheat; 4) Research was conducted into Al tolerance mechanisms in related cereal species (wheat, barley, maize, sorghum, rice), in order to see if they employ different tolerance mechanisms. It was found that these species employ a similar mechanism based on organic acid release from the root tip, which differed only in specific details such as the type of organic acid released (malate or citrate), the root region releasing the organic acid, and whether the response was constitutive or induced by Al exposure. These findings are important, for they suggest that similar or related genes regulate Al tolerance in each of these cereal crop species.
We have made significant progress in understanding a key mechanism that allows crop plants to grow on the acidic, aluminum-toxic soils that limit crop production on up to 50% of the world's soils. This mechanisms, which involves aluminum activation of the release of aluminum binding organic acids from roots, is helping direct our research aimed at isolating genes that confer crop aluminum tolerance. The isolation of aluminum tolerance genes will provide the molecular tools for improving aluminum tolerance in many crop species via biotechnology approaches.
- DiMuzio, E.M. 2000. Characterization of Or, a gene causing increased beta-carotene accumulation in cauliflower. M.S. Thesis. Cornell University. 71 p.
- Ebbs, S., Kochian, L.V., Lasat, M., Pence, N., Jiang, T. 2000. An integrated investigation of the phytoremediation of heavy metal and radionuclide contaminated soils: From the laboratory to the field. Wise, D.L., Trantolo, D.J., Inyang, H.I., Cichon, E.J., editors. Marcel Dekker, Inc., New York, NY. Remediation of Hazardous Wastes and Contaminated Soils. 2nd Edition. p. 745-769.
- Pence, N.S., Larsen, P.B., Ebbs, S.D., Lasat, M.M., Letham, D.L.D., Garvin, D.F., Eide, D., Kochian, L.V. 2000. The molecular basis for heavy metal hyperaccumulation in Thlaspi caerulescens. Proc. Natl. Acad.Sci USA.97:4956-4960.
- Cakmak, I., Welch, R.M., Hart, J.J., Norvell, W.A., Ozturk, L., Kochian, L.V. 2000. Uptake and re-translocation of leaf-applied cadmium (109Cd) in diploid, tetraploid and hexaploid wheats. J.Exp. Bot.51:221-226.
- Hoekenga, O.E., Kochian, L.V., Howell, S.D. 2000. Molecular genetic analysis of aluminum tolerance in arabidopsis thaliana. Plant Physiol. Abstracts. p. 98.
- Pence, N.S., Letham, D.L.D., Lasat, M.L., Jiang, T., Kochian, L.V. 2000. Altered Zn-dependent gene regulation in Thlaspi caerulescens plays a key role in heavy metal hyperaccumulation. Plant Physiol. Abstracts. p. 153
- Shaff, J.E., Tomos, D., Jones, D.L., Darrah, P., Kochian, L.V. 2000. Do roots of aluminum tolerant plants release enough organic acids to account for the observed Al tolerance? Plant Physiol. Abstracts. p. 93
- Wnag, Y-H., Kochian, L.V., Garvin, D.F. 2000. Mineral nutrition genomics: Expression profiling of genes involved in tomato plant mineral status and acquisition. Plant Physiol. Abstracts. p. 136.
- Kochian, L.V., Letham, D.L.D., Pence, N.S., Lasat, M.L., Jiang, T. 2000. The molecular physiology of heavy metal accumulation and tolerance in a metal hyperaccumulating plant species. Plant Physiol. Abstracts. p. 17.
- Cakmak, I., Welch, R.M., Erenoglu, B., Romheld, V., Norvell, W.A., Kochian, L.V. Influence of varied zinc supply on re-translocation of cadmium (109Cd) and rubidium (86RB) applied on mature leaf of durum wheat seedlings. 2000. Plant Soil 219:279-284.
- Tang, Y., Sorrells, M.E., Kochian, L.V., Garvin, D.F. 2000. Identification of RFLP markers linked to the barley aluminum tolerance gene Alp. Crop Sci. 40:778-782.
- Ebbs, S.D., Lau, I., Ahner, B.A., Kochian, L.V. 2000. Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Plant Physiol. Abstracts. p. 14.
- Hacisalihoglu, G,, Hart, J.J., Kochian, L.V. 2000. Investigation of the kinetics of root 65Zn uptake in Zn efficient and inefficient wheat (Triticum aestivum L.) genotypes reveal the existence of both high and low affinity Zn transport systems. Plant Physiol.Abstracts. p. 68.
- Hart, J.J., Welch, R.M., Norvell, W.A., Kochian, L.V. 2000. Correlating thiol and cadmium concentrations in tissues of near isogenic lines of durum wheat that differ in grain cadmium accumulation. Plant Physiol. Abstracts. p. 156.
- Letham, D.L.D., Pence, N.S., Pineros, M.A., Papoyan, A., Kochian, L.V. 2000. Molecular characterization of Zn/Cd uptake in the hyperaccumulator Thlaspi caerulescens including in situ RT-PCR localization and characterization of the heavy metal transporter ZNT1. Plant Physiol. Abstracts. p. 156.
- Mason, P.A., Garvin, D.F., Kochian, L.V. 2000. Isolating anion channel genes involved in aluminum tolerance in cereal crops. Plant Physiol. Abstracts. p. 97
Progress 01/01/99 to 12/31/99
This is a new project that began this year. We are working on two major subprojects. The first is addressing physiological and molecular mechanisms of aluminum tolerance in cultivated grass species, including maize, sorghum, wheat, barley and rice. The second project deals with the molecular physiology of heavy metal tolerance and accumulation in a unique hyperaccumulating plant species. As these are new projects, we don't have progress to report on for this year.
- No publications reported this period