Location: Plant, Soil and Nutrition Research2018 Annual Report
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.
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.
1. 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.
2. 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.
3. 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.
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