Progress 06/28/13 to 06/03/18
Outputs
Progress Report Objectives (from AD-416): 1: Determine mechanisms underlying the regulation of the major sorghum aluminum (Al) resistance gene, SbMATE, at the level of protein function, with the long term goal of identifying molecular determinants that interact with SbMATE to confer high levels of sorghum Al resistance. 1.1: Verification of SbMBP as an Al sensor and an Al-controlled switch for the SbMATE root citrate transporter. 1.2: Functional analysis of SbMBP and SbMATE proteins and their interactions. 1.3: Other protein-protein interactions modulating citrate transport mediated by SbMATE (and orthologues) 2: Conduct structure-function studies on members of a major family of cereal Al resistance proteins, the Multidrug and Toxic Compound Efflux (MATE) family of transporters, that function as root organic acid efflux transporters, to identify protein domains that play a role in conferring high levels of Al resistance. 2.1: Validation of structural and functional motifs that underlie key plant MATE transport properties. 2.2: Determination of the high-resolution structure of SbMATE by x- ray crystallography. 3: Identify and determine the roles of QTL and genes underlying these QTL identified from joint linkage/genome-wide association analysis for rice Al resistance and determine how gene-level variation influences rice Al resistance. 3.1: Fine scale map and clone the large effect rice Al resistance QTL identified on chr 12 from both bi- parental QTL mapping and GWA analysis. 3.2: Investigate the role of sequence variation for the candidate gene underlying a major QTL in the aus subpopulation, Nrat1, which encodes a rice root Al uptake transporter and determine the role this variation plays in aus Al resistance. 4: Investigate the genetic/genomic regulation of root system architecture (RSA) and the role of variation in RSA in rice and sorghum adaptation to nutrient�limited soils. 4.1: Mine the data from recently conducted joint linkage-GWA on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions. 4.2: Complete the development of a hydroponic-based system for investigating RSA in our sorghum association panel and complete GWA analysis of sorghum RSA traits in this panel. 5: Accelerate the adaptation of high throughput 3-D root imaging and image analysis to enhance the capacity of crops to adapt to climate change, increase water use efficiency, and improve nutrient use efficiency, through the genetic improvement of root architecture and physiology. Approach (from AD-416): 1) Study the role of sorghum AlMBP in regulating aluminum (Al) activated citrate transport via the sorghum Al tolerance protein, SbMATE. Will use a combination of ESI-Q-TOF MS/ ion mobility spectrometry and metal-ion chromatography to determine kinetics and specificity of Al binding by AlMBP. 2) Determine if Al binding by AlMBP causes this protein to disassociate from SbMATE using in vitro pull down assays, in vivo BiFC assays, and chemical cross-linking followed by LC-MS/MS analysis. 3) Determine the functional role of the SbMBP-SbMATE interaction by expressing both proteins in heterologous systems (oocytes and yeast) to determine if this confers Al activated of citrate exudation.4) Study the role of phosphorylation in regulation of SbMATE transport function via electrophysiological analysis of citrate efflux based on co-expression of SbMATE and candidate kinase proteins (CIPKs and calcineurin B-like [CBL] proteins) in oocytes.5) Investigate the role of protein structure in transport function for the plant MATE proteins that mediate citrate efflux and are involved in Al tolerance. Will determine the 3D crystal structure of SbMATE and use this structural model to direct functional analysis of SbMATE transport in oocytes. 6) After identifying altered SbMATE-type transporters that show enhanced function, the effects of these variants in plants will be determined by expressing SbMATE variants in transgenic Arabidopsis seedlings, and determining changes in Al tolerance. 7) In studies on rice Al tolerance, we will mine genome-wide association (GWA) data to identify/test candidate rice Al tolerance genes by a combination of high resolution mapping, molecular analysis in rice, expression of candidate Al tolerance genes in transgenic rice, and functional analysis of candidate transporter genes such as the Nrat1 Al transporter in heterologous systems (oocytes and yeast). 8) For research on root system architecture, we will mine data from joint linkage-GWA analysis on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions. This will involve a combination of fine scale mapping, mRNA seq analysis of candidate genes, expression of candidate RSA trait genes in transgenic rice, and the verification of functionality of different root architectures by looking at performance in soil under limiting (low water, N or P) conditions. This is the final report for 8062-21000-036-00D, �Genomic and Genetic Analysis of Crop Adaptation to Soil Abiotic Stresses,� which terminated in June 2018. In acidic soils aluminum (Al) ions readily solubilize from the clay minerals and become highly toxic to plant roots, damaging and stunting the root systems. Given the worldwide distribution of these type of soils (comprising about 40% of the world�s arable land including significant areas in the U.S. and in developing countries), Al toxicity is a major limitation to crop production. Using a multidisciplinary approach we made substantial progress across the four objectives and sub- objectives, gaining an in-depth understanding of the physiology and molecular nature of the mechanism used by crop plants, particularly sorghum and rice, to efficiently adapt to soil-based stress conditions. At the cellular level, we identified new members of diverse families of membrane transporter proteins that mediate the movement of toxic free aluminum, or organic molecules capable of chelating or binding this metal, thereby reducing its toxicity as it becomes immobilized, in turn enabling the plant�s root system to grow normally. We have investigated the relationship between the transport functions and structures of these proteins across multiple crop species. This in silico and experimentally validated approach has allowed us to identify common and conserved structural motifs in the proteins that underlie their ability to selectively transport organic acids. In addition, we identified accessory interacting proteins that regulate not only the expression of genes encoding these transporters but also their transport activity. In sorghum, we identified and extensively characterized an accessory protein that tightly binds to the transport protein SbMATE, thereby modulating its ability to transport organic acids out of the root, allowing release of sufficient organic acid to detoxify Al in the soil, while limiting the release of valuable carbon needed for plant growth. Using Arabidopsis as a model plant system, we have discovered an additional regulatory pathway serving to modify the organic acid transporter to reduce its transport activity. We also established the importance of the spatial distribution of the aluminum resistance response, by demonstrating that the protein associated with different molecular mechanisms and metabolic pathways are represented differentially in distinct cell types isolated from the same complex tissue. This cell type specificity is key to understanding mechanisms of aluminum resistance and how they can be exploited. Taken together, these cellular and molecular findings are significant, as they provide us with novel networks and molecular targets to be engineered or improved via molecular breeding for greater levels of Al tolerance and improving yields in important staple food crops. As part of understanding how plants adapt to various stresses in the soil, we were interested in understanding the basis of how plants place/ distribute the different root types throughout the soil, as root distribution has been shown to play a key role in improving the performance under both drought and low mineral nutrient conditions. For this purpose, we developed a variety of digital imaging platforms that allowed us to image root system architechure (RSA) of individual plants, and quantify nearly 20 individual traits contributing or related to RSA. These phenotyping platforms were used to conduct genome-wide association analysis of RSA traits of rice and sorghum diversity panels and genetically map individual RSA characters. We have identified regions in the rice and sorghum genome associated with RSA traits, as well as enhanced growth performance under suboptimal growth conditions (for example low soil P (phosphorus)). This information will be used to determine the role of RSA in important crop traits of rice and sorghum underlying efficient resource utilization (e.g. P and water), that can be used by plant breeders to develop higher yielding cereal varieties based on superior root traits. The results from this project provided an integrated understanding of the processes underlying abiotic stress responses at the molecular, cellular and organismal levels. The replacement project, �Genetic and Genomic Characterization of Crop Resistance to Soil-based Abiotic Stresses� builds on this acquired knowledge to continue to enhance our understanding on processes and specific molecular targets for enhancing agricultural productivity and sustainability through improved crop resilience and yield on marginal soils. Accomplishments 01 Soils high in aluminum which can inhibit plant growth and reduce crop performance. ARS researchers in Ithaca, New York, have identified novel transport proteins involved in the relocation of toxic aluminum (Al) within the plant, thereby unveiling a mechanism underlying genetic tolerance that can be utilized by breeders to develop crops that will better perform on high Al soils. Researchers have also identified accessory proteins and the chemical mechanism by which these systems work in two of the most important U.S. crops, corn, and sorghum. This new knowledge increases our understanding of tolerance responses and provides a set of new targets for breeders to use for improving yields on marginal soils, thus enhancing agricultural productivity and sustainability. 02 Understanding how interacting proteins regulate tolerance to Al stress. The sorghum SbMATE protein is a membrane transporter mediating the movement of organic acids that bind and immobilize toxic aluminum (Al), thereby providing Al resistance. SbMBP is an accessory protein that physically interacts with SbMATE, thereby regulating SbMATE transport. ARS researchers in Ithaca, New York, have used fluorescence-based assays to probe and characterized the interactions among various forms of the SbMATE and SbMBP proteins which were structurally modified. The observed changes in the characteristics of the interactions, relative to those observed among the unmodified SbMATE-SbMBP allowed the researchers to identify the structural regions in these two proteins which coordinate their physical interaction. This information enhanced our understanding on how these proteins functionally coordinate to promote Al tolerance while minimizing metabolic cost. Application of this knowledge has potential for enhancing crop yields on acid soils. 03 Protein identification at the level of individual cell type to resolve plant responses to Al toxicity. The physiological and molecular responses that take place during abiotic stress vary spatially throughout the plant. This variation is not only among different plant organs and tissues as expected but is also likely to occur among different and adjacent cells from a given tissue. Laser capture microdissection (LCM) is a microscopic technique capable of isolating small numbers of cells from a plant tissue for subsequent molecular or biochemical analysis. Since 2015 ARS scientists in Ithaca, New York, have systematically developed protocols that enable the identification of proteins that have been isolated from defined cells from the different regions of tomato roots. Although the initial results suggested different cell types within a tissue do respond differently to a particular stress, the number of cells that could be reasonably isolated and characterized was too small to support a proper quantitate analysis. The researchers have extensively optimized the LCM experimental processes increasing the number of cell capture by 8-10 fold, thereby allowing the identification and quantification of more than 7,000 proteins from a single experiment. The successful application of this new technology will facilitate and enhance our understanding of the biological processes involved in plant adaptation, providing knowledge that can be used to improve agriculture in marginal lands, such as acid soils.
Impacts
(N/A)
Publications
- Ligaba, A., Fei, Z., Liu, J., Xu, Y., Jia, X., Shaff, J., Lee, S., Luan, S. , Kudla, J., Kochian, L.V., Pineros, M. 2017. Loss-of-function mutation of the calcium sensor CBL1 increases aluminum sensitivity in Arabidopsis. New Phytologist. 214(2):830-841.
- Cheprot, R.K., Matonyei, T.K., Liu, J., Were, B.A., Dangasuk, G.O., Onkware, A.O., Ouma, E.O., Gudo, S., Kochian, L.V. 2013. Phylogenetic relationship among Kenyan sorghum germplasms based on aluminum tolerance. African Journal of Biotechnology. 12(22):3528-3536.
- Wang, Y., Cai, Y., Cao, Y., Liu, J. 2018. Aluminum-activated root malate and citrate exudation is independent of NIP1;2-facilitated root-cell-wall aluminum removal in Arabidopsis. Plant Signaling and Behavior. 13(1).
- Jiang, F., Wang, T., Wang, Y., Kochian, L., Chen, F., Liu, J. 2017. Identification and characterization of suppressor mutants of stop1. Biomed Central (BMC) Plant Biology. 17:128.
- Zhou, D., Yang, Y., Zhang, J., Jiang, F., Jia, X., Craft, E.J., Thannhauser, T.W., Kochian, L.V., Liu, J. 2017. Quantitative iTRAQ proteomics revealed possible roles for antioxidant proteins in sorghum aluminum tolerance. Journal of Proteome Research. 7:2043.
- Sangireddy, S., Ye, Z., Bhatti, S., Pei, X., Barozai, M., Thannhauser, T.W. , Zhou, S. 2017. Proteins in phytohormone signaling pathways for abiotic stress in plants. In: Pandey, P.K., editor. Mechanism of Plant Hormone Signaling Under Stress. Hoboken, New Jersey:John Wiley & Sons, Inc. p.187- 198.
- Rangu, M., Ye, Z., Bhatti, S., Zhou, S., Fish, T., Yang, Y., Thannhauser, T.W. 2018. Association of proteomics changes with Al-sensitive root zones in switchgrass. Proteomes. 6(2).
- Boldrin, P.F., Figueiredo, M., Yang, Y., Luo, H., Giri, S., Hart, J.J., Faquin, V., Guilherme, L., Thannhauser, T.W., Li, L. 2016. Selenium promotes sulfur accumulation and plant growth in wheat (Triticum aestivum). Physiologia Plantarum. 158:80-91.
- Doshim, R., McGrath, A., Pineros, M., Szewczyk, P., Garza, D., Kochian, L., Chang, G. 2017. Functional characterization and discovery of modulators of SbMATE, the agronomically important aluminium tolerance transporter from Sorghum bicolor. Scientific Reports. 7(17996).
- Liu, M., Lou, H., Chen, W., Pineros, M., Xu, J., Fan, W., Kochian, L., Yang, J., Zheng, S. 2018. Two citrate transporters coordinately regulate citrate secretion from rice bean root tip under aluminum stress. Plant, Cell & Environment. 41:809-822.
|
Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): 1: Determine mechanisms underlying the regulation of the major sorghum aluminum (Al) resistance gene, SbMATE, at the level of protein function, with the long term goal of identifying molecular determinants that interact with SbMATE to confer high levels of sorghum Al resistance. 1.1: Verification of SbMBP as an Al sensor and an Al-controlled switch for the SbMATE root citrate transporter. 1.2: Functional analysis of SbMBP and SbMATE proteins and their interactions. 1.3: Other protein-protein interactions modulating citrate transport mediated by SbMATE (and orthologues) 2: Conduct structure-function studies on members of a major family of cereal Al resistance proteins, the Multidrug and Toxic Compound Efflux (MATE) family of transporters, that function as root organic acid efflux transporters, to identify protein domains that play a role in conferring high levels of Al resistance. 2.1: Validation of structural and functional motifs that underlie key plant MATE transport properties. 2.2: Determination of the high-resolution structure of SbMATE by x- ray crystallography. 3: Identify and determine the roles of QTL and genes underlying these QTL identified from joint linkage/genome-wide association analysis for rice Al resistance and determine how gene-level variation influences rice Al resistance. 3.1: Fine scale map and clone the large effect rice Al resistance QTL identified on chr 12 from both bi- parental QTL mapping and GWA analysis. 3.2: Investigate the role of sequence variation for the candidate gene underlying a major QTL in the aus subpopulation, Nrat1, which encodes a rice root Al uptake transporter and determine the role this variation plays in aus Al resistance. 4: Investigate the genetic/genomic regulation of root system architecture (RSA) and the role of variation in RSA in rice and sorghum adaptation to nutrient�limited soils. 4.1: Mine the data from recently conducted joint linkage-GWA on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions. 4.2: Complete the development of a hydroponic-based system for investigating RSA in our sorghum association panel and complete GWA analysis of sorghum RSA traits in this panel. 5: Accelerate the adaptation of high throughput 3-D root imaging and image analysis to enhance the capacity of crops to adapt to climate change, increase water use efficiency, and improve nutrient use efficiency, through the genetic improvement of root architecture and physiology. Approach (from AD-416): 1) Study the role of sorghum AlMBP in regulating aluminum (Al) activated citrate transport via the sorghum Al tolerance protein, SbMATE. Will use a combination of ESI-Q-TOF MS/ ion mobility spectrometry and metal-ion chromatography to determine kinetics and specificity of Al binding by AlMBP. 2) Determine if Al binding by AlMBP causes this protein to disassociate from SbMATE using in vitro pull down assays, in vivo BiFC assays, and chemical cross-linking followed by LC-MS/MS analysis. 3) Determine the functional role of the SbMBP-SbMATE interaction by expressing both proteins in heterologous systems (oocytes and yeast) to determine if this confers Al activated of citrate exudation.4) Study the role of phosphorylation in regulation of SbMATE transport function via electrophysiological analysis of citrate efflux based on co-expression of SbMATE and candidate kinase proteins (CIPKs and calcineurin B-like [CBL] proteins) in oocytes.5) Investigate the role of protein structure in transport function for the plant MATE proteins that mediate citrate efflux and are involved in Al tolerance. Will determine the 3D crystal structure of SbMATE and use this structural model to direct functional analysis of SbMATE transport in oocytes. 6) After identifying altered SbMATE-type transporters that show enhanced function, the effects of these variants in plants will be determined by expressing SbMATE variants in transgenic Arabidopsis seedlings, and determining changes in Al tolerance. 7) In studies on rice Al tolerance, we will mine genome-wide association (GWA) data to identify/test candidate rice Al tolerance genes by a combination of high resolution mapping, molecular analysis in rice, expression of candidate Al tolerance genes in transgenic rice, and functional analysis of candidate transporter genes such as the Nrat1 Al transporter in heterologous systems (oocytes and yeast). 8) For research on root system architecture, we will mine data from joint linkage-GWA analysis on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions. This will involve a combination of fine scale mapping, mRNA seq analysis of candidate genes, expression of candidate RSA trait genes in transgenic rice, and the verification of functionality of different root architectures by looking at performance in soil under limiting (low water, N or P) conditions. During the fiscal year 2017, we have continued to make progress by meeting or exceeding the research goals in two separate but interlaced areas: 1) A significant part of the team�s research is aimed at understanding the physiological and genetic mechanisms underlying cereal crop adaptation to acid soils which comprise almost half of the world�s arable regions (including significant areas in the North East of the U.S. and developing countries). In this type of soil, aluminum ions are solubilized from clay minerals as phytotoxic free Al3+, which damages the root, generating a stunted the root systems which ultimately limits world crop productivity Understanding the basis of Al resistance mechanisms will provide a practical platform to exploit this knowledge to generate crops adapted to acid soils. 2) Research aimed at understanding the role and the genetic components governing the architecture of the root system as a means to improve plant performance and ultimately increase crop yields in marginal soils. With regards to plant aluminum tolerance research, we have extended our work identifying novel membrane transport proteins involved in mediating Al-resistance responses, as well as characterizing various accessory proteins that modulate the expression, as well as the activity of membrane transport proteins that underlie Al-resistance mechanisms: i) a novel transport protein, member of the aquaporin family, was shown to mediate the transport of the Al-malate complex from the root cell wall into the root symplasm, with subsequent Al translocation to the shoot. The activity of this transporter underlies a critical step in internal Al tolerance in Arabidopsis. ii) the novel sorghum protein SbMBP (for sorghum bicolor metal binding protein) is an aluminum (Al3+ ion) high-affinity binding protein, which binds preferentially Al3+ over other metal ions, in a pH dependent manner. SbMBP was shown to associates tightly with the SbMATE transport protein which facilitates citric acid efflux from roots into the rhizosphere where the citric acid binds and detoxifies Al3+ ions. Al3+ binding to SbMBP leads to changes in the conformation of SbMBP, resulting in disruption of the SbMATE-SbMBP interaction. Loss of this interaction results in SbMATE mediated citric acid transport out of the root, thereby activating and mediating Al-resistance. Using a series of deletion constructs of SbMBP and SbMATE, we have identified candidate interaction domains of SbMBP and SbMATE required for their interactions. We have sequenced the DNA sequences for the SbMATE and SbMBP genes from hundreds of different sorghum lines in our sorghum diversity panel. We have identified several different classes of DNA sequence variants for each gene, analyzed the correlation of specific DNA sequences with Al tolerance and root organic acid exudation. We are continuing to examine the relationship between genetic variation (differences in DNA sequence) and differences in protein function relating to sorghum Al tolerance. iii) through a comparative physiological and whole transcriptome investigation with have stablished that calcineurin B-like calcium sensors (CBLs) and CBL-interacting protein kinases (CIPKs) are involved in Al resistance responses. We have successfully identified a CBL/CIPK complex that interacts with AtMATE (an orthologue protein to SbMATE) and modulates its citrate transport activity. This protein complex constitutes part of a calcium -regulated pathway involved in the Al- resistance response, by which phosphorylation of the downstream target protein, AtMATE1, limits unnecessary carbon loss via unregulated citrate exudation, thereby regulating the abiotic stress tolerance response in a temporal and spatial manner. iv) We have continued to improve proteomic technologies to advance crop Al tolerance research. The root tip proteomes of several plant species were screened in response to control and Al toxicity treatments using multiplexed isotope coding technology. The goal of these experiments is to identify Al responsive proteins that may underlie novel Al tolerance mechanisms. The number of Al responsive proteins identified varied by species, experimental condition, and developmental stage. Previously we reported on the enhanced activity of multiple antioxidant enzymes upon Al stress in a Al tolerant sorghum line (relative to the unchanged levels observed in Al- sensitive line). The list of Al responsive proteins is being investigated further in these species to determine their biological function through literature mining and homology to proteins of known function. We have continued to refine and develop existing and new root phenotyping platforms for studying root system architecture (RSA) of crop plants. We are interested in understanding the mechanisms determining how plants place/distribute the different root types throughout the soil, as this has been shown to play a key role in improving the performance under both drought and low mineral nutrient conditions. We have developed a new phenotyping approach in which individual seedling are grown in a pouch system that allows us to maintain the root spatial characteristics. Implementation of this new platform is allowing us to increase the throughput, thereby imaging a larger number of plants in less time, a feature which is essential for genetic studies. Accomplishments 01 Many soils in the U.S. and parts of the world are naturally high in aluminum (Al) which can inhibit plant growth and reduce crop performance. ARS researchers in Ithaca, New York have identified novel transport proteins involved in the relocation of toxic Al within the plant, thereby unveiling a mechanism underlying genetic tolerance that can be utilized by breeders to develop crops that will better perform on high Al soils. Researchers have also identified accessory proteins and the chemical mechanism by which these systems work in two of the most important U.S. crops, corn and sorghum. This new knowledge increases our understanding of the complexity of the tolerance responses and provides a set of new targets for breeders to use for improving yields on marginal soils, thus enhancing agricultural productivity and sustainability. 02 Protein identification at the level of individual cell type to resolve plant responses to Al toxicity. Laser capture microdissection (LCM) is a microscopic technique that isolates small numbers of cells from a plant tissue for subsequent molecular or biochemical analysis. ARS scientists in Ithaca, New York, have dramatically improved a workflow originally reported in 2015 to identify proteins from cells isolated from the different regions of tomato roots. The original work suggested that distinct cell types within a specific tissue respond differently to particular biotic and abiotic challenges and that these responses must be integrated into a coordinated global response. However, the number of cells that could be reasonably isolated using the original protocol was too small to support the quantitate analysis needed to understand the biological processes involved in adaptation to growth on acid soils. The improved protocol increases the rate of cell capture by 3-4 fold making quantitative analysis using isobaric, isotope coded mass tags possible for the first time. 03 Second generation plant root imaging and data acquisition system. ARS researchers in Ithaca, New York have developed an improved design to collect digital images (2D & 3D) of the root systems of a range of agricultural crops. There is a growing need to measure both older and larger root systems to improve our understanding of the genetics that control favourable rooting traits underlying crop productivity, for future implementation in plant breeding programs. A larger imaging apparatus with a simplified control system using an innovative plant root growth media was prototyped, along with the release of new operating and imaging software. The new design presents a simplified means for collecting, managing and preserving root system images for future analysis. The new system has made data acquisition efficient by reducing imaging time, increasing system capacity, and providing safe storage of the critical experimental data.
Impacts
(N/A)
Publications
- Martin, L., Scherwood, R., Nicklay, J., Yang, Y., Muratore, S., Anderson, E., Thannhauser, T.W., Rose, J., Zhang, S. 2016. Application of wide selected-ion monitoring data-independent acquisition to identify tomato fruit proteins regulated by the CUTIN DEFICIENT2 transcription factor. Proteomics. 16:2081-2094.
- Zhu, Y., Hui, L., Bhatti, S., Zhou, S., Yang, Y., Fish, T., Thannhauser, T. W. 2016. Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots. Horticulture Research. 3:16026.
- Zhou, S., Okekeogbu, I., Sangireddy, S., Yi, Z., Hui, L., Bhatti, S., Hui, D., Yang, Y., Howe, K.J., Fish, T., Thannhauser, T.W. 2016. Proteome modification in tomato plants upon long-term aluminum treatment. Journal of Proteome Research. 15:1670-1684.
- Ye, Z., Sangireddy, S., Okekeogbu, I., Zhou, S., Yu, C., Hui, D., Howe, K. J., Fish, T., Thannhauser, T.W. 2016. Drought-induced leaf proteome changes in switchgrass seedlings. International Journal of Molecular Sciences. 17:1251-1269.
- Yuan, H., Owsiang, K., Sheeja, T., Zhou, X., Rodriguez, C., Li, Y., Welsch, R., Chayut, N., Yang, Y., Thannhauser, T.W., Pathasarathy, M.V., Xu, Q., Deng, X., Fei, Z., Schaffer, A., Katzir, N., Burger, J., Tadmor, Y., Li, L. 2014. A single amino acid substitution in an ORANGE protein promotes carotenoid overaccumulation in arabidopsis. Plant Physiology. 169(1):421- 431.
|
Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): 1: Determine mechanisms underlying the regulation of the major sorghum aluminum (Al) resistance gene, SbMATE, at the level of protein function, with the long term goal of identifying molecular determinants that interact with SbMATE to confer high levels of sorghum Al resistance. 1.1: Verification of SbMBP as an Al sensor and an Al-controlled switch for the SbMATE root citrate transporter. 1.2: Functional analysis of SbMBP and SbMATE proteins and their interactions. 1.3: Other protein-protein interactions modulating citrate transport mediated by SbMATE (and orthologues) 2: Conduct structure-function studies on members of a major family of cereal Al resistance proteins, the Multidrug and Toxic Compound Efflux (MATE) family of transporters, that function as root organic acid efflux transporters, to identify protein domains that play a role in conferring high levels of Al resistance. 2.1: Validation of structural and functional motifs that underlie key plant MATE transport properties. 2.2: Determination of the high-resolution structure of SbMATE by x- ray crystallography. 3: Identify and determine the roles of QTL and genes underlying these QTL identified from joint linkage/genome-wide association analysis for rice Al resistance and determine how gene-level variation influences rice Al resistance. 3.1: Fine scale map and clone the large effect rice Al resistance QTL identified on chr 12 from both bi- parental QTL mapping and GWA analysis. 3.2: Investigate the role of sequence variation for the candidate gene underlying a major QTL in the aus subpopulation, Nrat1, which encodes a rice root Al uptake transporter and determine the role this variation plays in aus Al resistance. 4: Investigate the genetic/genomic regulation of root system architecture (RSA) and the role of variation in RSA in rice and sorghum adaptation to nutrient�limited soils. 4.1: Mine the data from recently conducted joint linkage-GWA on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions. 4.2: Complete the development of a hydroponic-based system for investigating RSA in our sorghum association panel and complete GWA analysis of sorghum RSA traits in this panel. 5: Accelerate the adaptation of high throughput 3-D root imaging and image analysis to enhance the capacity of crops to adapt to climate change, increase water use efficiency, and improve nutrient use efficiency, through the genetic improvement of root architecture and physiology. Approach (from AD-416): 1) Study the role of sorghum AlMBP in regulating aluminum (Al) activated citrate transport via the sorghum Al tolerance protein, SbMATE. Will use a combination of ESI-Q-TOF MS/ ion mobility spectrometry and metal-ion chromatography to determine kinetics and specificity of Al binding by AlMBP. 2) Determine if Al binding by AlMBP causes this protein to disassociate from SbMATE using in vitro pull down assays, in vivo BiFC assays, and chemical cross-linking followed by LC-MS/MS analysis. 3) Determine the functional role of the SbMBP-SbMATE interaction by expressing both proteins in heterologous systems (oocytes and yeast) to determine if this confers Al activated of citrate exudation.4) Study the role of phosphorylation in regulation of SbMATE transport function via electrophysiological analysis of citrate efflux based on co-expression of SbMATE and candidate kinase proteins (CIPKs and calcineurin B-like [CBL] proteins) in oocytes.5) Investigate the role of protein structure in transport function for the plant MATE proteins that mediate citrate efflux and are involved in Al tolerance. Will determine the 3D crystal structure of SbMATE and use this structural model to direct functional analysis of SbMATE transport in oocytes. 6) After identifying altered SbMATE-type transporters that show enhanced function, the effects of these variants in plants will be determined by expressing SbMATE variants in transgenic Arabidopsis seedlings, and determining changes in Al tolerance. 7) In studies on rice Al tolerance, we will mine genome-wide association (GWA) data to identify/test candidate rice Al tolerance genes by a combination of high resolution mapping, molecular analysis in rice, expression of candidate Al tolerance genes in transgenic rice, and functional analysis of candidate transporter genes such as the Nrat1 Al transporter in heterologous systems (oocytes and yeast). 8) For research on root system architecture, we will mine data from joint linkage-GWA analysis on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions. This will involve a combination of fine scale mapping, mRNA seq analysis of candidate genes, expression of candidate RSA trait genes in transgenic rice, and the verification of functionality of different root architectures by looking at performance in soil under limiting (low water, N or P) conditions. During fiscal year 2016 we have continued to make progress by meeting or exceeding the research goals. The research progress in fiscal year 2016 has continued in three separate but interlaced areas: 1) A significant part of the team�s research is aimed at understanding the physiological and genetic mechanisms underlying cereal crop adaptation to acid soils comprising almost half of the world�s arable regions. In this type of soil, including significant areas in the U.S. and in developing countries, aluminum ions are solubilized from clay minerals as phyto-toxic free Al3+, damaging and stunting the root systems, posing a major limitation to world crop production. Therefore, the understanding of Al tolerance mechanisms will provide a platform to use this knowledge to generate crops adapted to acid soils, 2) Research aimed at understanding the role and the genetic components governing the architecture of the root system as a means to improve plant performance and ultimately increase crop yields in marginal soils, and 3) Development of new protein-based research technologies. With regards to plant Al tolerance research, we have demonstrated that a novel sorghum protein we previously identified, SbMBP (for sorghum bicolor metal binding protein) is an aluminum (Al3+ ion) binding protein and we have shown it binds Al3+ ions with very high affinity in preference to other related metal ions (La, Fe, Mg) in a pH dependent manner. We have also demonstrated that SbMBP associates tightly with the major sorghum Al tolerance protein SbMATE, which facilitates citric acid efflux from roots into the rhizosphere where the citric acid binds and detoxifies Al3+ ions. When SbMBP binds Al3+, the SbMATE-SbMBP interaction is disrupted because the Al binding changes the conformation of SbMBP, which reduces the strength of the SbMATE-SbMBP interaction. When this happens, the SbMATE transporter can transport citric acid out of the root. We also have sequenced the DNA sequences for the SbMATE and SbMBP genes from hundreds of different sorghum lines in our sorghum diversity panel. We have identified several different classes of DNA sequence variants for each gene, analyzed the correlation of specific classes of the DNA sequences with Al tolerance and root organic acid exudation. We will be looking at the relationship between differences in genetic variation (differences in DNA sequence) and differences in protein function relating to sorghum Al tolerance. We also have used improvements in proteomic technologies to advance crop Al tolerance research. The root tip proteomes of several plant species (sorghum, rice, Arabidopsis) were screened in response to control and Al toxicity treatments, in Al sensitive and tolerant cultivars and over a range of developmental stages, using multiplexed isotope coding technology. The goal of these experiments was to identify Al responsive proteins that may underlie novel Al tolerance mechanisms. Proteome coverage was high (> 5,000 proteins quantified/experiment) and number of Al responsive proteins identified varied by species, experimental condition and developmental stage. In the case of sorghum, the activities of multiple antioxidant enzymes (SOD, POD, and CAT etc.) were enhanced by Al stress in a tolerant line (SC566) while they remained unchanged or suppressed in a sensitive variety (BR007). Reactive oxygen species (ROS) are known to promote lignin formation and since lignin may be an Al binding substrate in the cell wall, higher levels of lignin accumulation in root tips of BR007 could lead to higher levels of Al retained in this region, leading to the accumulation of higher levels of Al and increased toxicity to the root tips. The list of Al responsive proteins is being investigated further in these species to determine their biological function through literature mining and homology to proteins of known function. We have continued to refine and develop existing and new root phenotyping platforms for studying root system architecture (RSA) of crop plants. We are interested in understanding the mechanisms determining how plants place/distribute the different root types throughout the soil, as this has been shown to play a key role in improving the performance under both drought and low mineral nutrient conditions. We continue to work in collaboration with researchers from the University of Nottingham using X- ray computed tomography to image root systems in soil and assemble those images into 3D reconstructions of the RSA. We are currently in the process of developing and validating a new approach in which individual seedling are grown in a pouch system under hydroponic conditions, while still maintaining their root spatial characteristics. Implementation of this will allow us to increase the throughput, allowing us to image a larger number of plants in less time, a feature which is essential for genetic studies. Accomplishments 01 Protein identification at the level of individual cell type to resolve plant responses to Al toxicity. LCM is a microscopic technique that isolates small numbers of cells from a plant tissue for subsequent molecular or biochemical analysis. ARS scientists in Ithaca, New York, have used a workflow reported in 2015 to identify proteins from cells isolated from the different regions of tomato roots. It was found that Al-stress-related proteins, such as metal handling proteins, ferritin and several proteins belonging to the ABC transporters and multidrug resistance systems, are observed in epidermal but not cortical cells. This suggests that distinct cell types within a specific tissue respond differently to particular biological challenges and that these responses must be integrated into a coordinated global response. Such an understanding may prove useful in the generation of tomato and other crop plants that are better adapted to growth on acid soils. 02 Identification of accessory proteins regulating the function of plant Al tolerance transport proteins. ARS researchers in Ithaca, New York have identified the mechanism by which various proteins bind to and regulate the function of a previously discovered Al tolerance protein in corn and sorghum. The tolerance proteins are involved in transporting organic acids (OA�s) from the root into the Al-toxic soil, where the OA�s detoxify the Al ions by binding to them. The accessory regulatory proteins alter both the localization and the ability of the transport protein to release OAs into the soil when no Al is near the root, thereby minimizing the energetic cost associated with releasing OAs. This new knowledge increases our understanding of the complexity of the tolerance responses, and provides a set of new targets for breeding programs to be used via molecular breeding with the aim of improving the energy (carbon) efficiency of the Al tolerance based on root OA release, thereby improving yields on acid soils. 03 Second Generation Plant Root Imaging and Data Acquisition System. ARS researchers at the Robert W. Holley Center in Ithaca, New York have developed an improved design to collect digital images (2D & 3D) of the root systems of a range of agricultural crops. There is a growing need to measure both older and larger root systems to improve our understanding of the genetics that control favorable rooting traits for plant breeding programs to combat the risks presented by climate change. A larger imaging apparatus with a simplified control system using an innovative plant root growth media has been prototyped. New operating software has been released. The new design presents a simplified means for collecting, managing and preserving root system images for future analysis. The new system has made data acquisition efficient by reducing imaging time, increasing system capacity, and providing safe storage of the critical experimental data.
Impacts
(N/A)
Publications
- Pineros, M., Larson, B., Shaff, J., Schneider, D.J., Falcao, A., Lixing, Y. , Clark, R.T., Craft, E.J., Davis, T.W., Pradier, P., Liu, J., Assaranurak, I., Susan, M., Sturrock, C., Bennett, M., Kochian, L.V. 2016. Advances and considerations in technologies for growing, imaging, and analyzing 3-D root system architecture. Journal of Integrative Plant Biology. 58(3):230- 241.
- Brauer, E.K., Ahsan, N., Dale, R., Kato, N., Coluccio, A.E., Pineros, M., Kochian, L.V., Thelen, J.J., Popescu, S. 2016. The raf-like kinase ILK1 and the high affinity K+ transporter HAK5 are required for innate immunity and abiotic stress response. Plant Physiology. doi: 10.1104/pp.16.00035.
- Zhang, Z., Zheng, Y., Ham, B., Chen, J., Yoshida, A., Kochian, L.V., Fei, Z., Lucas, B. 2016. Plant vasculature-mediated signaling involved in early phosphate stress response. Nature Plants. doi: 10.1038/nplants.2016.33.
- Coskun, D., Britto, D.T., Kochian, L.V., Kronzucker, H.J. 2015. How high do ion fluxes go? A re-evaluation of the two-mechanism model of K+ 2 transport in plant roots. Plant Science. 243:96-104.
- Hart, J.J., Tako, E.N., Kochian, L.V., Glahn, R.P. 2015. Identification of black bean (Phaseolus vulgaris L.) polyphenols that inhibit and promote iron uptake by caco-2 cells. Journal of Agricultural and Food Chemistry. DOI: 10.1021/acs.jafc.5b00531.
- Zhou, X., Welsch, R., Yang, Y., Riediger, M., Alvarez, D., Yuan, H., Fish, T., Liu, J., Thannhauser, T.W., Li, L. 2015. Arabidopsis OR proteins are the major post-transcriptional regulators of phytoene synthase in mediating carotenoid biosynthesis. Proceedings of the National Academy of Sciences. 112:3558-3563.
|
Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): 1: Determine mechanisms underlying the regulation of the major sorghum aluminum (Al) resistance gene, SbMATE, at the level of protein function, with the long term goal of identifying molecular determinants that interact with SbMATE to confer high levels of sorghum Al resistance. 1.1: Verification of SbMBP as an Al sensor and an Al-controlled switch for the SbMATE root citrate transporter. 1.2: Functional analysis of SbMBP and SbMATE proteins and their interactions. 1.3: Other protein-protein interactions modulating citrate transport mediated by SbMATE (and orthologues) 2: Conduct structure-function studies on members of a major family of cereal Al resistance proteins, the Multidrug and Toxic Compound Efflux (MATE) family of transporters, that function as root organic acid efflux transporters, to identify protein domains that play a role in conferring high levels of Al resistance. 2.1: Validation of structural and functional motifs that underlie key plant MATE transport properties. 2.2: Determination of the high-resolution structure of SbMATE by x- ray crystallography. 3: Identify and determine the roles of QTL and genes underlying these QTL identified from joint linkage/genome-wide association analysis for rice Al resistance and determine how gene-level variation influences rice Al resistance. 3.1: Fine scale map and clone the large effect rice Al resistance QTL identified on chr 12 from both bi- parental QTL mapping and GWA analysis. 3.2: Investigate the role of sequence variation for the candidate gene underlying a major QTL in the aus subpopulation, Nrat1, which encodes a rice root Al uptake transporter and determine the role this variation plays in aus Al resistance. 4: Investigate the genetic/genomic regulation of root system architecture (RSA) and the role of variation in RSA in rice and sorghum adaptation to nutrient�limited soils. 4.1: Mine the data from recently conducted joint linkage-GWA on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions. 4.2: Complete the development of a hydroponic-based system for investigating RSA in our sorghum association panel and complete GWA analysis of sorghum RSA traits in this panel. 5: Accelerate the adaptation of high throughput 3-D root imaging and image analysis to enhance the capacity of crops to adapt to climate change, increase water use efficiency, and improve nutrient use efficiency, through the genetic improvement of root architecture and physiology. Approach (from AD-416): 1) Study the role of sorghum AlMBP in regulating aluminum (Al) activated citrate transport via the sorghum Al tolerance protein, SbMATE. Will use a combination of ESI-Q-TOF MS/ ion mobility spectrometry and metal-ion chromatography to determine kinetics and specificity of Al binding by AlMBP. 2) Determine if Al binding by AlMBP causes this protein to disassociate from SbMATE using in vitro pull down assays, in vivo BiFC assays, and chemical cross-linking followed by LC-MS/MS analysis. 3) Determine the functional role of the SbMBP-SbMATE interaction by expressing both proteins in heterologous systems (oocytes and yeast) to determine if this confers Al activated of citrate exudation.4) Study the role of phosphorylation in regulation of SbMATE transport function via electrophysiological analysis of citrate efflux based on co-expression of SbMATE and candidate kinase proteins (CIPKs and calcineurin B-like [CBL] proteins) in oocytes.5) Investigate the role of protein structure in transport function for the plant MATE proteins that mediate citrate efflux and are involved in Al tolerance. Will determine the 3D crystal structure of SbMATE and use this structural model to direct functional analysis of SbMATE transport in oocytes. 6) After identifying altered SbMATE-type transporters that show enhanced function, the effects of these variants in plants will be determined by expressing SbMATE variants in transgenic Arabidopsis seedlings, and determining changes in Al tolerance. 7) In studies on rice Al tolerance, we will mine genome-wide association (GWA) data to identify/test candidate rice Al tolerance genes by a combination of high resolution mapping, molecular analysis in rice, expression of candidate Al tolerance genes in transgenic rice, and functional analysis of candidate transporter genes such as the Nrat1 Al transporter in heterologous systems (oocytes and yeast). 8) For research on root system architecture, we will mine data from joint linkage-GWA analysis on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions. This will involve a combination of fine scale mapping, mRNA seq analysis of candidate genes, expression of candidate RSA trait genes in transgenic rice, and the verification of functionality of different root architectures by looking at performance in soil under limiting (low water, N or P) conditions. The project has continued to make good progress on all goals and is meeting or exceeding all significant research goals for FY15. The research progress is in three areas: crop aluminum tolerance, development of new protein-based research technologies and research into the role of root system architecture in improving crop yields on less fertile soils � �how to get crops to do more with less water and fertilizer.� Much of the team�s research is on cereal crop adaptation to acid soils. Acid soils comprise 40% of the world�s soil, including significant areas in the U.S. and in developing countries. On acid soils, Al ions are solubilized from clay minerals and are highly toxic to plant roots, damaging and stunting the root systems. Because these acid soils are so extensive worldwide, Al toxicity is a major limitation to world crop production and there is significant research going on to identify Al tolerance genes and mechanisms, and to use this information to generate crops adapted to acid soils. With regards to plant Al tolerance research, we have demonstrated that a novel sorghum protein we previously identified, SbMBP (for sorghum bicolor metal binding protein) is an aluminum (Al3+ ion) binding protein and we have shown it binds Al3+ ions with very high affinity in preference over other related metal ions (La, Fe, Mg). We have also demonstrated that SbMBP associates tightly with the major sorghum Al tolerance protein SbMATE, which facilitates citric acid efflux from roots into the rhizosphere where the citric acid binds and detoxifies Al3+ ions. When SbMBP binds Al3+, the SbMATE-SbMBP interaction is disrupted because the Al binding changes the conformation of SbMBP, which reduces the strength of the SbMATE-SbMBP interaction. When this happens, the SbMATE transporter can transport citric acid out of the root. We also have sequenced the DNA sequences for the SbMATE and SbMBP genes from hundreds of different sorghum lines in our sorghum diversity panel. We have identified several different classes of DNA sequence variants for each gene and will be looking at the relationship between differences in genetic variation (differences in DNA sequence) and differences in protein function relating to sorghum Al tolerance. We also have used recent improvements in proteomic technologies in our group to advance our crop Al tolerance research. The root tip proteomes of several plant species (sorghum, rice, A. thaliana) were screened in response to control and Al toxicity treatments, in Al sensitive and tolerant cultivars and over a range of developmental stages, using multiplexed isotope coding technology. The goal of these experiments was to identify Al responsive proteins that may underlie novel Al tolerance mechanisms. Proteome coverage was high, with more than 5,000 proteins quantified in each experiment. The number of Al responsive proteins identified varied by species, experimental condition and developmental stage of the plant. The list of Al responsive proteins is currently being investigated to determine their biological function through literature mining and homology to proteins of known function. Al responsive proteins whose assigned function suggest plausible involvement in processes that can enhance Al tolerance will be validated using targeted quantitative approaches and other orthogonal means. We have also continued to advance and improve new methods for studying root system architecture (RSA) of crop plants. We are interested in how plants control the 3D placement of the different root types in the soil as this has been shown to play a key role in improved performance under drought and low mineral nutrient conditions. We have been working in collaboration with researchers from the University of Nottingham using X- ray computed tomography to image root systems in soil and reconstruct those images into 3D reconstructions of RSA. We discovered a very useful growth substrate that mimics the soil and allows for more rapid and detailed imaging of rice, sorghum and corn roots. The roots of intact plants were grown in a specially prepared soil amendment, known as Turface, which are calcine clay particles that have been baked at 1500 degree F. The soil amendment is a nice mimic for soil, but allows X-ray imaging of roots to proceed much more quickly, allowing us to image many plants (which is needed for genetic studies). Using this system, we have obtained very accurate reconstructions of the root systems of 15 day old rice plants. This is opening up new avenues for phenotyping roots and quantifying RSA traits of plants grown in soil-like media with enough throughputs to conduct sophisticated genetic mapping studies of RSA traits. Accomplishments 01 Coupling Laser Capture Micro-dissection (LCM) and protein identification to resolve plant cellular responses to aluminum (Al) toxicity. Acid soils are widespread in the United States and worldwide, and crop yields on acid soils are low because aluminum ions are dissolved from clay minerals at low soil pH, and are toxic to plant roots. Hence there is intense interest in identifying crop Al tolerance genes to use to improve yields on acid soils. The LCM technique is a microscopic technique that isolates small numbers of cells from a plant tissue for subsequent molecular or biochemical analysis. ARS scientists in Ithaca, New York, working with colleagues at Tennessee State University, have developed a novel workflow which greatly improves protein extraction from small LCM samples of plant roots and was to identify approx. 1,300 proteins from cells isolated from the different regions of tomato roots. Importantly, it was found that Al stress induced the production of enzymes that repair DNA presumably in response to Al damage of DNA sequence. This demonstrates that even with a small sample size that contains a relatively small number of highly expressed proteins, important biological differences can be found, and gives valuable information that could be used in the long term to generate tomato and crop plants adapted to acid soils. 02 Identification of accessory proteins regulating the function of plant aluminum (Al) tolerance proteins. Acid soils comprise 40% of the world�s soil and 20% of the soils in the U.S. Crop yields on acid soils are low because aluminum (Al) ions are dissolved from clay minerals at low soil pH, and are toxic to plant roots. Hence, there is intense interest in identifying crop Al tolerance genes and proteins to use via molecular breeding to improve yields on acid soils. ARS researchers in Ithaca, New York, have identified novel proteins that bind to and regulate the function of a previously discovered Al tolerance protein in corn and sorghum. The tolerance protein mediates Al-activated release of organic acids (OA�s) from the root into the Al-toxic soil, where the OA�s detoxify the Al ions. The accessory proteins function to phosphorylate the Al tolerance protein in the root, inhibiting its ability to transport OA�s into the soil when no Al is near the root. Because OA release is energetically costly to the plant, it is important for this process to be finely regulated so it only occurs when the root is exposed to toxic Al ions. This knowledge enhances our understanding of new targets for breeding for crops with more energy (carbon) efficient Al tolerance based on root OA release, thus improving yields on acid soils. 03 Identification of an aquaporin membrane transporter involved in root-to- shoot aluminum ion translocation. Acid soils comprise 40% of the world�s soil and 20% of the soils in the U.S. Crop yields on acid soils are low because aluminum (Al) ions are dissolved from clay minerals at low soil pH, and are toxic to plant roots. Hence, there is intense interest in identifying crop Al tolerance genes and proteins to use via molecular breeding to improve yields on acid soils. ARS researchers in Ithaca, New York, are studying a novel mechanisms of crop Al tolerance involving the transport of Al that has entered the root to the shoot, where Al ions are sequestered in leaf cell vacuoles. However, the mechanisms by which Al ions are transported to the shoot were unknown. In Arabidopsis plants, a novel aquaporin transporter gene was identified. Aquaporins are membrane transporters that were believed to transport just water and neutral solutes (such as glycerol) across membranes. Here researchers showed this unique aquaporin transports Al ions form the root to the shoot, reducing the level of toxic Al in sensitive root tissues. It appears that this aquaporin transporter plays a role in a new mechanism of plant Al tolerance and in the future may be a new target for plant breeders to develop crop varieties with increased yields on Al toxic acid soils.
Impacts
(N/A)
Publications
- Milner, M., Mitani-Ueno, N., Yamaji, N., Yokosho, K., Craft, E.J., Fei, Z., Ebbs, S., Ma, J., Kochian, L.V. 2014. Root and shoot transcriptome analysis of two ecotypes of Noccaea caerulescens uncovers the role of NcNramp1 in Cd hyperaccumulation. Plant Journal. 78:398-410.
- Hufnagel, B., De Sousa, S.M., Assis, L., Guimaraes, C.T., Leiser, W., Corradi, G., Negri, B., Larson, B.G., Shaff, J.E., Pastina, M., Barros, B. A., Weltzien, E., Rattunde, H.W., Viana, J.H., Clark, R.T., Falcao, A., Gazaffi, R., Garcia, A.F., Schaffert, R.E., Kochian, L.V., Magalhaes, J.V. 2014. Duplicate and conquer: multiple homologs of phosphorus-starvation tolerance 1 enhance phosphorus acquisition and sorghum performance on low- P soils. Plant Physiology. 166(2):659-677.
- Tako, E.N., Hoekenga, O., Kochian, L.V., Glahn, R.P. 2013. High bioavailable iron maize (Zea mays L.) developed through molecular breeding provides more absorbable iron in vitro and in vivo. Nutrition Journal. 12:3.
- Li, J., Liu, J., Dong, D., Jia, X., Mccouch, S., Kochian, L.V. 2014. Natural variation underlies alterations in NRAT1 expression and function that play a key role in rice aluminum tolerance. Proceedings of the National Academy of Sciences. 111(17):6503-6508.
- Thannhauser, T.W., Shen, M., Sherwood, R., Howe, K.J., Fish, T., Yang, Y., Chen, W., Zhang, S. 2013. A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry. Electrophoresis. DOI: 10. 1002/elps.201200656.
- Avila, F., Yang, Y., Faquin, V., Ramos, S., Guilherme, L., Thannhauser, T. W., Li, L. 2014. Impact of selenium supply on se-methylselenocysteine and glucosinolates accumulation in selenium-biofortified brassica sprouts. Food Chemistry. 165:578-586.
- Ruiz-May, E., Hucko, S., Howe, K.J., Zhang, S., Sherwood, R.W., Thannhauser, T.W., Rose, J.K. 2014. A comparative study of lectin affinity based plant n-glycoproteome profiling using tomato fruit as a model. Proteomes. 13:566-579.
- Zhou, S., Palmer, M., Zhou, J., Bhatti, S., Howe, K.J., Fish, T., Thannhauser, T.W. 2013. Differential root proteome expression in tomato genotypes with contrasting drought tolerance exposed to dehydration. Journal of the American Society for Horticultural Science. 138(2):131-141.
- Pinheiro, P., Bereman, M., Burd, J., Pals, M.A., Armstrong, J.S., Howe, K. J., Thannhauser, T.W., Maccoss, M., Gray, S.M., Cilia, M. 2014. Evidence for the biochemical basis of host virulence in the greenbug aphid, Schizaphis graminum (Homoptera: Aphididae). Journal of Proteome Research. 13(4):2094�2108.
- Okekeogbu, I., Ye, Z., Sangireddy, S.R., Li, H., Bhatti, S., Zhou, S., Howe, K.J., Fish, T., Yang, Y., Thannhauser, T.W. 2014. Effect of aluminum treatment on proteomes of radicles of seeds derived from Al-treated tomato plants. Proteomes. 2(2):169-190.
- Kochian, L.V., Pineros, M., Liu, J., Magalhaes, J. 2015. Plant adaptation to acid soils: the molecular basis for crop aluminum resistance. Annual Reviews of Plant Biology. 66:571-598. DOI: 10.1146/annualrev-arplant- 043014-114822.
- Caniato, F.F., Hamlin, M.T., Guimaraes, C.T., Zhang, Z., Schaffert, R.E., Kochian, L.V., Magalhaes, J.V. 2014. Association mapping provides insights into the origin and the fine structure of the sorghum aluminum tolerance locus, AltSB. PLoS One. 9(1):e87438.
- Matonyei, T., Cheprot, R., Liu, J., Pineros, M., Shaff, J., Gudo, S., Were, B., Magalhaes, J., Kochian, L.V. 2014. Physiological and molecular analysis of selected Kenyan maize lines for aluminum tolerance. Plant Journal. 377:357-367.
|
Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): 1: Determine mechanisms underlying the regulation of the major sorghum aluminum (Al) resistance gene, SbMATE, at the level of protein function, with the long term goal of identifying molecular determinants that interact with SbMATE to confer high levels of sorghum Al resistance. 1.1: Verification of SbMBP as an Al sensor and an Al-controlled switch for the SbMATE root citrate transporter. 1.2: Functional analysis of SbMBP and SbMATE proteins and their interactions. 1.3: Other protein-protein interactions modulating citrate transport mediated by SbMATE (and orthologues) 2: Conduct structure-function studies on members of a major family of cereal Al resistance proteins, the Multidrug and Toxic Compound Efflux (MATE) family of transporters, that function as root organic acid efflux transporters, to identify protein domains that play a role in conferring high levels of Al resistance. 2.1: Validation of structural and functional motifs that underlie key plant MATE transport properties. 2.2: Determination of the high-resolution structure of SbMATE by x- ray crystallography. 3: Identify and determine the roles of QTL and genes underlying these QTL identified from joint linkage/genome-wide association analysis for rice Al resistance and determine how gene-level variation influences rice Al resistance. 3.1: Fine scale map and clone the large effect rice Al resistance QTL identified on chr 12 from both bi- parental QTL mapping and GWA analysis. 3.2: Investigate the role of sequence variation for the candidate gene underlying a major QTL in the aus subpopulation, Nrat1, which encodes a rice root Al uptake transporter and determine the role this variation plays in aus Al resistance. 4: Investigate the genetic/genomic regulation of root system architecture (RSA) and the role of variation in RSA in rice and sorghum adaptation to nutrient�limited soils. 4.1: Mine the data from recently conducted joint linkage-GWA on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions. 4.2: Complete the development of a hydroponic-based system for investigating RSA in our sorghum association panel and complete GWA analysis of sorghum RSA traits in this panel. 5: Accelerate the adaptation of high throughput 3-D root imaging and image analysis to enhance the capacity of crops to adapt to climate change, increase water use efficiency, and improve nutrient use efficiency, through the genetic improvement of root architecture and physiology. Approach (from AD-416): 1) Study the role of sorghum AlMBP in regulating aluminum (Al) activated citrate transport via the sorghum Al tolerance protein, SbMATE. Will use a combination of ESI-Q-TOF MS/ ion mobility spectrometry and metal-ion chromatography to determine kinetics and specificity of Al binding by AlMBP. 2) Determine if Al binding by AlMBP causes this protein to disassociate from SbMATE using in vitro pull down assays, in vivo BiFC assays, and chemical cross-linking followed by LC-MS/MS analysis. 3) Determine the functional role of the SbMBP-SbMATE interaction by expressing both proteins in heterologous systems (oocytes and yeast) to determine if this confers Al activated of citrate exudation.4) Study the role of phosphorylation in regulation of SbMATE transport function via electrophysiological analysis of citrate efflux based on co-expression of SbMATE and candidate kinase proteins (CIPKs and calcineurin B-like [CBL] proteins) in oocytes.5) Investigate the role of protein structure in transport function for the plant MATE proteins that mediate citrate efflux and are involved in Al tolerance. Will determine the 3D crystal structure of SbMATE and use this structural model to direct functional analysis of SbMATE transport in oocytes. 6) After identifying altered SbMATE-type transporters that show enhanced function, the effects of these variants in plants will be determined by expressing SbMATE variants in transgenic Arabidopsis seedlings, and determining changes in Al tolerance. 7) In studies on rice Al tolerance, we will mine genome-wide association (GWA) data to identify/test candidate rice Al tolerance genes by a combination of high resolution mapping, molecular analysis in rice, expression of candidate Al tolerance genes in transgenic rice, and functional analysis of candidate transporter genes such as the Nrat1 Al transporter in heterologous systems (oocytes and yeast). 8) For research on root system architecture, we will mine data from joint linkage-GWA analysis on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions. This will involve a combination of fine scale mapping, mRNA seq analysis of candidate genes, expression of candidate RSA trait genes in transgenic rice, and the verification of functionality of different root architectures by looking at performance in soil under limiting (low water, N or P) conditions. In the first year of this project, ARS researchers at Ithaca, New York, have built upon their discovery of a sorghum gene (SbMATE) that confers tolerance to aluminum (Al) toxicity on acid soils that comprise 40% of the world�s soils and over 20% of US soils. SbMATE encodes a protein that transports citric acid from the growing roots into the acid soil where it binds and detoxifies Al ions that can damage and inhibit root systems. This allows the roots to grow in the toxic environment. We now have identified and verified the functioning of a novel mechanism of regulation of SbMATE. A second protein, SbMBP (SbMATE binding protein) was identified that binds very strongly to the SbMATE protein and regulates its function. The binding of SbMBP to SbMATE blocks citrate transport. SbMBP is an Al sensor and when it binds Al ions, it is released from the SbMATE protein, allowing the transport of citrate out of the root. This regulation of SbMATE ensures that there is no wasteful carbon loss from the root as citrate is very important for many plant processes. ARS researchers in Ithaca, NY have also studied other modes of regulation of the SbMATE protein that mediates citrate release from the root. One major mode of regulation of protein function is a process by which a phosphate group is added to specific amino acids in the protein by a second protein known as a protein kinase, a process known as phosphorylation. Once the target protein is phosphorylated, this can alter its function. Researchers identified two novel kinase proteins that phosphorylate the SbMATE protein, turning off its ability to transport citrate. This appears to be another important switch controlling the release of citrate that is essential for detoxifying Al3+ ions in the soil but this occurs at a cost to the plant, as citrate is a very important carbon resource for the plant. Hence, we are discovering how important regulation of this function is for carbon efficient aluminum tolerance. ARS researchers in Ithaca, NY have been developing new methods for studying root system architecture (RSA) of crop plants which plays a key role in improved crop performance on nutrient and water limited soils. Researchers have been growing roots in different media (gel, hydroponics, glass beads, sand, permanent clay particles) to study crop plant�s root system architecture under conditions which are moving closer to the �real world� - plants growing in the field. The root systems were grown in clay particles (turface) that mimic soil and the roots grow through a specially designed plastic mesh system that maintains the 3D architecture of the root systems. The turface growth system is promising, as the clay particles maintain their structure even in water. We have grown maize plants in the plastic mesh system in pots containing turface watered with nutrient solution. After 10 days, we were able to separate the pot from the growing plant, immerse the roots, mesh and turface in water and the turface readily washes away from the roots. It was then possible to image the root system and reconstruct the 3D root system architecture in roots that were grown in a relatively realistic growth medium. Significant Activities that Support Special Target Populations: ARS researchers at Ithaca, NY have hosted two summer undergraduate interns from Clemson University and Ohio Wesleyan University. The students participated as part of a summer program between the USDA, Boyce Thompson Institute, and Cornell University funded by the National Science Foundation to involve under-represented minorities and women in plant science research. The students worked on a project with ARS scientists on the role of aquaporins in Arabidopsis Al tolerance described above in the progress report and on a project on salinity tolerance in rice over a 10 week period. Each undergraduate then presented a 15 minute talk on the work at a symposium held at the end of their research training. During this period of time, the students learned useful skills in molecular biology, root phenotyping, hydroponic culture of plants, and whole plant physiology, which enhanced their technical background, scientific knowledge, and problem solving skills. ARS researchers at Ithaca, NY are also hosting graduate students from the Department of Agricultural Sciences at Tennessee State University, an 1890 College with a tradition of educating under representative minorities in the sciences. The visitors are supported by an AFRI Strengthening Grant and are working with ARS scientists continuously for their entire stay. Accomplishments 01 Identification of plant transport proteins that underlie a novel plant trait that enables the plant to tolerate toxic aluminum (Al) ions. Acid soils comprise 40% of the world�s soils, including significant areas in the US and in developing countries. On acid soils Al ions are solubilized from clay minerals and are highly toxic to plant roots, damaging and stunting the root systems. Because of these acid soils are so extensive worldwide, Al toxicity is a major limitation to crop production. ARS researchers at Ithaca, New York studied different plant transport proteins to identify those that can transport Al ions. They identified a class of transporters, aquaporins that transport Al ions from the soil into the root. They showed that these transport proteins underlie a novel Al tolerance mechanism. This discovery is significant, for these researchers can now modify these aquaporins in major crop plants such as corn and sorghum to improve their Al tolerance and thus improve crop yields on acid soils that are second only to drought worldwide as abiotic stresses that limit crop yields. 02 Investigating the molecular basis for high expression of plant aluminum (Al) tolerance genes that is important for greater crop Al tolerance. Acid soils comprise 40% of the world�s soils, including significant areas in the US and in developing countries. On acid soils Al ions are solubilized from clay minerals and are highly toxic to plant roots, damaging and stunting the root systems. Because of these acid soils are so extensive worldwide, Al toxicity is a major limitation to crop production. ARS researchers at Ithaca, New York have recently cloned a number of genes in corn, sorghum, rice and the model experimental plant species, Arabidopsis that confer Al tolerance. In each case they have studied different varieties within each species that are either Al tolerant or sensitive, and have found that high expression of Al tolerance genes is key to elevated Al tolerance in each plant species. Thus they have employed unique genetic approaches to identify other plant genes that play a role in the molecular processes controlling elevated gene expression of these tolerance genes. They are currently working with different versions of these 'accessory' genes to use them to increase expression of Al tolerance genes in crop plant varieties that normally have low expression of these genes. This discovery is significant, for gaining the ability to increase the expression of Al tolerance genes in major crop plants such as corn and sorghum will improve crop Al tolerance and thus improve crop yields on acid soils that are second only to drought worldwide as abiotic stresses that limit crop yields. 03 Accumulation of organo-selenium compounds in food crops. Selenium (Se) is an essential nutrient for humans and also has been shown to have potent anticancer properties. ARS researchers at Ithaca, New York have studied of the impact of selenium supply on the synthesis of organo- selenium (Se-methylslenocysteine)and organic-sulfur (glucosinolates) compounds that play a role in preventing cancer in the six most consumed brassica species. It was found that biofortification with Se did not adversely impact the ability of Brassica plants to synthesize significant amounts of the organ0-sulfer compounds which was a concern as plants use the same pathways to incorporate sulfur and Se into organic compounds. Furthermore, analysis of the glucosinolate profiles of the species studied demonstrate that different Brassica species accumulate different types and amounts of glucosinolates and that cauliflower accumulates the highest amount of the potent anticancer glucosinolate compound, glucoraphanin. These findings indicate that biofortification with Se can be done without negative effects on the concentrations of these important chemopreventive compounds.
Impacts
(N/A)
Publications
- Suqin, F., Clark, R., Zheng, Y., Iyer-Pascuzzi, A.S., Weitz, J., Kochian, L.V., Edelsbrunner, H., Liao, H., Benfey, P. 2013. Genotypic recognition and spatial responses by rice roots. Proceedings of the National Academy of Sciences. 110(7):2670-2675.
- Souza, G., Hart, J., Carvalho, J., Rutzke, M., Albrecht, J., Guilherme, L., Kochian, L.V., Li, L. 2014. Genotypic variation of zinc and selenium content in grains of Brazilian wheat lines. Plant Science. 224:27-35.
- Schroeder, J.I., Delhaize, E., Frommer, W.B., Guerinot, M., Harrison, M.J., Herrera-Estrella, L., Horie, T., Kochian, L.V., Munns, R., Nishizawa, N.K. , Tsay, Y., Sanders, D. 2013. Using membrane transporters to improve crops for sustainable food production. Nature. 497:60-66.
- Milner, M., Pineros, M., Kochian, L.V. 2014. Molecular and physiological mechanisms of plant tolerance to toxic metals. In: Jenks, M.A. and Hasegawa, P.M., editors. Plant Abiotic Stress, Second Edition. Hoboken, NJ: John Wiley & Sons, Inc. p. 179-196.
- Liu, J., Pineros, M., Kochian, L.V. 2014. The role of aluminum sensing and signaling in plant aluminum resistance. Journal of Integrative Plant Biology. 56(3):221-230. DOI: 10.1111/jipb.12162
- Milner, M., Pence, N., Liu, J., Kochian, L.V. 2014. Identification of a novel pathway involving a GATA transcription factor in yeast and possibly plant Zn uptake and homeostasis. Journal of Integrative Plant Biology. 56(3):271-280. DOI: 10.1111/jipb.12169.
- Magalhaes, J.V., Maron, L.G., Pineros, M., Guimaraes, C.T., Kochian, L.V. 2013. Aluminum tolerance in sorghum and maize. In: Varshney, R., Ruberosa, R., editors. Translational genomics for crop breeding: improvement for abiotic stress, quality and yield improvement. Volume 2. Chichester, UK. Wiley and Sons Ltd. p. 83-98. DOI: 10.1002/9781118728482.ch6
- Guimaraes, C.T., Simoes, C.C., Lyza, M.G., Pastina, M.M., Magalhaes, J.V., Vasconcellos, R., Guimaraes, L., Lana, U., Tinoco, C., Noda, R.W., Jardim- Belicuas, S.N., Parentoni, S.N., Alves, V., Kochian, L.V. 2014. Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan. Biomed Central (BMC) Genomics. 15:153. DOI:10.1186/1471-2164-15-153.
- Lyi, J., Liu, J., Dong, D., Jia, X., Mccouch, S., Kochian, L.V. 2014. Natural variation underlies alterations in Nramp aluminum transporter (NRAT1) expression and function that play a key role in rice aluminum tolerance. Proceedings of the National Academy of Sciences. 111(17):6503- 6508.
- Ligaba, A., Dreyer, I., Margaryan, A., Schneider, D.J., Kochian, L.V., Pineros, M. 2013. Functional, structural and phylogenetic analysis of domains underlying the Al-sensitivity of the aluminium-activated malate/ anion transporter, TaALMT1. Plant Journal. 76(6):766-780. DOI: 10.1111/ tpj.12332
- Gayumba, S., Jung, H., Yan, J., Danku, J., Rutzke, M.A., Bernal, M., Kramer, U., Kochian, L.V., Salt, D.E., Vatamaniuk, O.K. 2013. The CTR/COPT- dependent copper uptake and SPL7-dependent copper deficiency responses are required for basal cadmium tolerance in A. thaliana. Metallomics. 5:1262- 1275. DOI: 10.1039/c3mt00111c
- Wang, X., Wang, Z., Pineros, M., Wang, Z., Wang, W., Li, C., Wu, Z., Kochian, L.V., Wu, P. 2014. Phosphate transporters OsPHT1;9 and OsPHT1;10 are involved in phosphate uptake in rice. Plant Cell and Environment. 37(5) :1159-1170. DOI: 10.1111/pce.201437.issue-5/issuetoc.
- Zhiyang, Z., Gayomba, S.R., Jung, H., Vimalakumar, N.K., Pineros, M., Rutzke, M., Danku, J., Lahner, B., Punshon, T., Guerinot, M., Salt, D.D., Kochian, L.V., Vatamaniuk, O.K. 2014. OPT3 is a phloem-specific iron transporter that is essential for systemic iron signaling and redistribution of iron and cadmium in arabidopsis. The Plant Cell. DOI: http:dx.doi.org/10.1105/tpc.114.123737.
|
|