Progress 09/01/07 to 08/31/10
Outputs
OUTPUTS: We have used the Two Electrode Voltage Clamp (TEVC) technique to determine and study the functional characteristics of ALMT-type proteins expressed in Xenopus oocytes and yiled the following results: 1. We studied the transport specificity of TaALMT1, and its orthologs AtALMT1 and ZmALMT1 and ZmALMT2, for orgabnic acid anion (malate and citrate) over inorganic anion (nitrate, sulfate, Cl)efflux. 2. Structural motifs underlying transport features. a) ALMT type proteins consist of an highly hydrophobic N-terminus region consisting of 5 to 7 membrane spanning domains, followed by a long hydrophilic C terminal tail which comprises about half of the protein. We systematically altered the structures of the ALMTs from wheat, Arabidopsis and maize and examined their transport properties, to identify protein's domains determining their unique functional characteristics. b) We had previous identified specific structural motifs in the C-terminus region involved in the modulation of TaALMT1 transport activity via PKC-mediated phosphorylation in response to Al. To investigate the role of the C-terminus in transport regulation and function, as well as its interactions with the pore forming region (i.e. N-terminus) we studied the functional characteristics of six protein chimeras where the N and C terminus regions were swapped among three distinct ALMT orthologues. c) We generated single point mutations in TaALMT1 for those residues (all 43 negatively charge residues in the TaALMT1 protein) presumed to be involved in Al3+ binding. Each of these residues was substituted by an uncharged polar amino acids (i.e. Asp to Asn, or Glu to Gln) and sensitivity (or lack of sensitivity) to extracellular Al3+ was examined. c)We used two techniques, Bimolecular Fluorescence Complementation (BiFC) and total internal reflection (TIR)-single-molecule photo-bleaching (SMPB) microscopy, to look at protein-protein interactions in ALMT-type proteins, to determine if functional ALMT proteins assemble as an aggregate of several subunits. The findings were disseminated as 3 publications in Plant Journal and Plant Physiology with 4 more manuscripts in prepration for submission to PNAS, Plant Journal and Plant Physiology, 5 published symposia proceedings, and 12 talks presented at major international and national meetings and symposia. Broad Impacts: TaALMT1 is the major wheat Al tolerance gene encoding a root malate efflux transporter Using molecular and electrophysiological approaches, we have significantly advanced our understanding of the relationship between ALMT protein structure and its function as an Al tolerance protein. This includes an understanding of structural features associated with Al activation/enhancement of malate efflux, and permeation selectivity that results in some ALMT proteins preferentially transporting malate over inorganic anions, while others transporting inorganic anions. This increased understanding should enable the identification and/or design the best ALMT alleles with regards to increased levels of Al tolerance for a range of crop plants grown on acid soils. PARTICIPANTS: Training: Dr. Ayalew Ligaba (Postdoctoral Associate) is the primary scientist working and being trained on the project. He has generated and characterized TaALMT1 truncated proteins. Dr. Ligaba has also developed and characterized the single point mutants used to establishe the role of protein phosphorylation in regulating TaALMT1 activity. He has written a major manuscript listed above that has been submitted to Plant Journal. Armine Margaryan (Ph.D candidate student from Yerevan State University in Armenia) worked between April to August 2008, learning and collaborating in the molecular and electrophysiological characterization of mutant generated towards identifying Al3+ binding sites. We anticipate she will be returning in 2010 to continue her training Collaborations: Dr. Kochian visited the Root Biology Institute at South China Agricultural University in Guangzhou, China, in 2009 where he presented a series of invited seminars including one on the work funded by the grant on ALMT1 transporters. This led to the development of a collaboration with Dr. Hong Liao, the Director of the Root Biology Institute. Dr. Liao will be sending a postdoctoral associate to Dr. Kochian's lab to use the TEVC/oocyte approach to study the transporter properties of ALMT and MATE transporters involved in soybean root organic acid efflux underlying both Al tolerance and phosphorus efficiency. Soybean is a major crop grown on huge expanses of acid soils in China. Our lab has established a new collaboration in 2009 with Dr. Isacoff of the Department of Molecular and Cell Biology at the University of California, Berkeley. Dr. Isacoff's lab has developed and successfully implemented the use of total internal reflection (TIR) and single-molecule photo-bleaching (SMPB) microscopy as means to determine the nature of protein oligomerization in vivo. We have been working closely with Mr. Ryan Arant, a PhD student in Dr Isacoff's lab, in the analysis of the oligomerization of TaALMT1, as well as the other ALMT orthologues. In 2009 we established a new collaboration with Dr. Geoffrey Chang of The Scripps Research Institute, La Jolla, CA, in an effort to complement our functional analysis of the ALMT transporters with his expertise in x-ray crystallography of transport proteins. The research described so far has allowed us to obtain a thorough understanding of the protein's functional features, which will provide the basis to correlate these features with the 3-D structural information gained from a high-resolution structure of TaALMT1 determined by x-ray crystallography. The integration of the functional (electrophysiological) and structural (x-ray crystallography) research will enable us in a future to identify and target those protein residues/motifs underlying TaALMT1 transport properties critical for Al tolerance. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
1) TaALMT1 was found to selectively mediate the efflux of malate, and had a lower permeabilty to inorganic anions (Cl, NO3 and SO42-).ZmALMT1, unlike TaALMT1 and AtALMT1, preferentially mediates efflux of inorganic anions over malate anions and is insensitive to extracellular Al3+. However, ZmALMT2 mediates malate and citrate efflux, but is insensitive to extracellular Al3+. 2) Functional analysis of truncated proteins generated by sequentially deleting segments of the C-terminus established that that in the absence of the long hydrophilic C terminal tail, the hydrophobic (N-terminus) of the protein harboring the membrane spanning domains remains functional as it is permeable to malate but insensitive to Al3+. This indicates the hydrophobic N-terminus contains the domains determining the permeability/selectivity properties of these transporters. 3) We investigated the role of the C-terminus in ALMT transport function and its interactions with the N terminal pore forming region, via functional characterization of six protein chimeras where the N and C terminus regions were swapped among three distinct ALMT orthologues (TaALMT1, AtALMT1, and ZmALMT1. Due to space limitations, we will summarie the findings for the chimeras here. The characterization of the transport properties of these 6 chimeras allowed us to determine that although the N-terminus half of the ALMT proteins (the pore region) forms a functionally active transporter capable of mediating transport of anions, the unique ion selectivity, and transport regulation (e.g., enhancement by extracellular Al3+) are the product of subtle interactions between the highly hydrophobic N and hydrophilic C termini of the protein. 4) Transport characterization of the site-directed mutagenesis of the 43 negative residues in TaALMT1 led to the identification of three specific single point mutations which entirely eliminate the transport enhancement by extracellular Al3+. About half of the remaining mutants showed reduced enhancement by Al3+. These findings allowed us to locate specific residues involved in interaction(s) between Al3+ and the TaALMT1 protein. Likewise, the fact that some of single point mutations showing lack of or reduced Al3+ sensitivity are located in the N-terminus of the protein, reinforce the nature of N and C termini interactions taking place during the conformational changes required for transport activity enhancement following Al3+ binding to the protein. 5) We have identified protein-protein interactions in ALMT-type proteins, demonstrating that functional ALMT proteins assemble as an aggregate of several subunits. The BIFC approach determined the existence of protein-protein interactions leading to multimer assembly of ALMT proteins when expressed in the mammalian cells, HEK293. Subsequently, the TIR-SMPB technique confirmed these interactions and determined that ALMTs assemble as a dimer. We are currently investigating the functional importance of this dimerization for malate efflux.
Publications
- 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.
- 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.
- 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.
- 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.
- Pineros MA, Cancado GMA, Maron LG, Sangbom ML, Menossi M and Kochian LV. 2008. Not all ALMT1-type transporters mediate aluminum-activated organic acid responses: The case of ZmALMT1. Plant Journal 53, 352-367.
- Pineros MA, Geraldo GMA and Kochian LV. 2008. Novel transport properties of TaALMT1 (aluminum-activated organic acid transporter) revealed by functional characterization in Xenopus oocytes: Functional and Structural implications. Plant Physiol. 147, 2131-2146
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: 1. We have characterized the regulation of TaALMT1, the malate efflux transporter which underlies the Aluminum (Al) tolerance response in wheat. Functional modulation of TaALMT1 transport activity by ligand (i.e. Al3+) binding and/or phosphorylation mediated events has been 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. These findings indicate that direct phosphorylation of the TaALMT1 transporter by protein kinases plays a role in modulating its transport activity. B) We have performed the 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, indicating that the hydrophobic N-terminus region contains the transmembrane domains underlying the pore forming region or the transporter, being the main determinant of the permeation properties of a given transporter chimera. In fact, chimeras containing only the N-terminal region are capable of mediating ion transport, regardless of the presence or absence of the C-terminal region. In contrast, the regulation (i.e. enhancement of transport activity) by extracellular Al3+ is not determined solely by the hydrophilic C-terminus regions, but rather by an interaction between this and the N terminus region. This inference is also substantiated by results where negatively charged amino acids, presumed to be involved in the enhancement of ALMT1 activity by Al3+ were substituted by uncharged polar amino acid (e.g. such as Asp to Asn or Glu to Gln), and functionally evaluated in X. oocytes. Out of the 43 negatively charge residues found through out the TaALMT1 protein, 30 single-point mutations have been evaluated. Of these only 3 amino acid substitutions on residues 95, 166 and 275 result in a loss of transport enhancement upon exposure to extracellular Al3+. The remaining substitutions do not result in functional alteration, or results in a slight loss of affinity to Al3+. These results, in conjunction with the swap domain chimera results, suggest that not only one, but several negatively charged residues through out the protein are involved in the binding (and subsequently enhancement of transport activity) of Al3+ to the protein. C) We have performed a comparative study between two closely related maize ALMT homologues (ZmALMT1 and ZmALMT2), examining their differences in permeability to different organic acids, with the aim at identifying potential residue candidates along the N-terminus regions involved in determining the permeability/selectivity transport properties 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. TARGET AUDIENCES: We have also used this grant to train an Eastern European/Western Asia student in molecular and electrophysiological techniques. Ms. Armine Margaryan is a Ph.D student from Yerevan State University in Armenia who came to work in our lab from April to August, 2008, learning and collaborating on the molecular and electrophysiological aspects of the project. She will be returning in early 2010 to continue her training by working on this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
TaALMT1 is the major Al tolerance gene in wheat that encodes a membrane transport protein that facilitates organic acid (malate anion) efflux from roots. Combining molecular and electrophysiological approaches we have increased our understanding of the characteristics that underlie the structural and functional properties of this transporter. We demonstrated that although the transport activity (i.e. organic acid efflux) of TaALMT1 is highly dependent and enhanced by the binding of extracellular Al3+ to the protein, the protein is also functionally active and can mediate anion transport in the absence of this ligand. The electrophysiological data indicates that the "enhancement" of TaALMT1 malate transport by Al is not due to an alteration in the transporter's selectivity properties, but solely due to increases in its 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. Analysis of truncated versions of TaALMT1, as well a chimeric proteins containing different domains from three ALMT orthologs indicates 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 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 have also identified specific amino acid residues which are required for Al3+ binding, and consequently enhancement of the transport activity of TaALMT. We have identified the presence of protein-protein interactions in TaALMT1, suggesting 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. Presentations: 1) "Structure-Function Analysis of ALMT Transporters Involved in Crop Aluminum Tolerance" at the COMBIO 2008 Conference, September 21-25, 2008, Canberra, Australia. 2) "Structure-Function Relations Underlying the Functionality of the ALMT and MATE-type Transporters Involved in the Organic Acid Release Al Tolerance Response". Annual meeting of the American Society of Plant Physiologists. July 2009, Hawaii, USA. 3) "Protein phosphorylation regulates the transport activity of the wheat TALMT1 aluminum tolerance protein". Annual meeting of the American Society of Plant Physiologists. July 2009, Hawaii, USA
Publications
- 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.
- Ligaba A, Kochian LV, Pineros MA. 2009. Modulation of TaALMT1 transport activity by protein phosphorylation. In: Plant-Soil Interactions at Low pH: A Nutriomic Approach. Proceedings of the 7th International Symposium on Plant-Soil Interactions at Low pH. Guangzhou, China. Eds, H. Liao, X Yan and LV Kochian. pp. 73-74.
- Pineros MA, Kochian LV. 2009. Overview of the structure-function relations underlying functionality of ALMT and MATE-type transporters involved in the organic acid release Al tolerance response. In: Plant-Soil Interactions at Low pH: A Nutriomic Approach. Proceedings of the 7th International Symposium on Plant-Soil Interactions at Low pH. Guangzhou, China. Eds, H. Liao, X Yan and LV Kochian. pp. 55-56.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: 1. In studying the transport properties of the malate efflux transporter, TaALMT1, that is also responsible for wheat Aluminum (Al) tolerance. we determined its permeability/selectivity and Al3+ binding affinity characteristics via Two Electrode Voltage Clamp (TEVC) in Xenopus oocytes expressing TaALMT1. 2. Determined the functional similarities/dissimilarities between TaALMT1 and its maize homologue, ZmALMT1, using TEVC in oocytes. 3. Determined the role of phosphorylation in TaALMT1 transport activity by evaluating changes in transport activity (via TEVC) after exposure to a wide range of kinase and phosphatase inhibitors. 4. Did preliminary work to study whether TaALMT1 and its homologs in maize (ZmALMT1) and Arabidopsis (AtALMT1) function as monomers or via multimeric complexes. Two different approaches are being employed: A) chimeras designed for Bimolecular Fluorescence Complementation (BiFC) to evaluate the existence of protein-protein interactions leading to multimer assembly of ALMT1 proteins, and B) chimeras designed for single-molecule photo-bleaching (SMPB) which will allow us to determine the number of subunits in putative channle protein multimeric complexes. Visualization of the fluorescence signal, as well as functional evaluation via TEVC from HEK293 and/or X. oocytes cells expressing these chimeras is currently being performed. 5. Several mutated and truncated versions of the TaALMT1 protein have been constructed to evaluate the effect of these structural changes on the functionality of the protein. These constructs include: A)sequential truncations of the hydrophilic C- terminus of TaALMT1; B) chimeras where the hydrophilic C- terminus and the hydrophobic N-terminus of the 3 ALMT1 homologues have been swapped. C) TaALMT1 constructs containing single point mutations modifying six predicted phosphorylation sites. D) Single point mutations in which every extracellular negatively charged residue has been substituted by an uncharged polar amino acid, to look for residues that bind Al3+. Changes in transport function are being assayed using electrophysiological approaches (TEVC) in X. oocytes expressing the various constructs. Our findings have been shared with the research community via the following presentations: "Crop Aluminum Tolerance: Molecular and Genetic Investigations of a Rhizosphere-Mediated Agronomic Trait", Plenary Address - Rhizosphere 2 Symposium, Montpellier, France, August 25-31, 2007. "Identification and characterization of novel aluminum tolerance genes in cereal crops", USA-Pakistan Symposium on Plant Stress Biology, University of California, Davis, November 4-5, 2007. "Isolation and Characterization of Aluminum Tolerance Genes in the Cereals at the Plant and Animal Genome XVI Meeting, 2008. "Aluminum-activated citrate and malate transporters encoded by distinct Al tolerance genes function independently in Arabidopsis". Root Genomics Workshop, Plant and Animal Genome XVI Meeting, 2008. "Positional Cloning and Association Analysis of a MATE Gene that Confers Aluminum Tolerance in Sorghum via the AltSB Locus. Sorghum and Millets 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 compentently trained over the last year in the implementation of electrophysiological techniques (e.g., two electrode voltage clamp) that will enable future expression and functional characterization of the various chimeras, mutations, and truncations of the ALMT-family of proteins. TARGET AUDIENCES: We have also used this grant to train an Eastern European/Western Asia student in molecular and electrophysiological techniques. Ms. Armine Margaryan is a Ph.D candidate student from Yerevan State University in Armenia who came to work in our lab from April to August, 2008, learning and collaborating in the molecular and electrophysiological aspects of the project. We anticipate she will be returning in 2009 to continue her training. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
TaALMT1 is the major Al tolerance gene in wheat that encodes a membrane transport protein that facilitates organic acid (malate anion)efflux from roots. Using electrophysiological techniques, we have recently increased our understanding of the functional properties of this transporter. We demonstrated that although the transport activity (i.e. organic acid efflux) of TaALMT1 is highly dependent and enhanced by the presence of extracellular Al3+ (presumably due to the binding of Al3+ to the protein), the protein is also functionally active and can mediate anion transport in the absence of extracellular Al3+. The electrophysiological data indicates that the "enhancement" of TaALMT1 malate transport by Al is not due to an alteration in the transporter's selectivity properties, but solely due to increases in its anion permeability. We have shown that TaALMT1 can selectively transport malate compared to other anions. In addition, this protein can also mediate a lesser transport of other physiologically relevant anions such as Cl-, NO3- and SO42-. We are currently studying 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. Analysis of truncated versions of TaALMT1 indicates 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 the C-terminus hydrophilic portion of the protein. The presence of the C-terminus (hydrophilic regions) is required for Al activation of malate efflux. We have pinpointed specific amino acid residues located in the C-terminus region which are involved in the Al3+ binding, and consequently enhancement of the transport activity of TaALMT. Additionally, preliminary results indicate the presence of protein-protein interactions in TaALMT1, suggesting 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.
Publications
- Pineros MA, Cancado GMA, Maron LG, Sangbom ML, Menossi M and Kochian LV (2008) Not all ALMT1-type transporters mediate aluminum-activated organic acid responses: The case of ZmALMT1. Plant Journal 53, 352
- Pineros MA, Geraldo GMA and Kochian LV (2008) Novel transport properties of TaALMT1 (aluminum-activated organic acid transporter) revealed by functional characterization in Xenopus oocytes: Functional and Structural implications. Plant Physiol. 147, 2131
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