Location: Plant, Soil and Nutrition Research
2023 Annual Report
Objectives
Objective 1: Analyze the structure and biochemical functions of selected ALMT, MATE, aquaporin (AQP), and Nramp membrane transporters in relation to Al tolerance and mineral nutrient deficiency to develop improved adaption to acid soil environments.
Objective 2: Identify the genes and molecular pathways that modulate the expression and activity of transporters that confer Al tolerance, including interacting proteins/complexes, as well as post translational modifications.
Objective 3: Dissect the signaling networks that control and regulate resistance to low pH and Al stress in Arabidopsis for ultimate application in cereal crop improvement.
Objective 4: Analyze the genetic control and the environmental regulation of root system architecture (RSA) and the role of variation in RSA in rice and sorghum adaptation to acid soils focusing on Al toxicity and P deficiency.
Objective 5: Analyze differential protein expression, at the cellular level in root tips, as a function of Al exposure at acidic pH, to understand specific tissue and cell type functions.
Approach
1) Identification of structural motifs that underlie key functional transport properties in the transporters associated with Al-resistance responses. We will express structurally altered transporters in heterologous systems and evaluate changes in their functionality via electrophysiological and fluxes analysis. Selected structural variants will be expressed in transgenic Arabidopsis seedling to determine their effect on the plant Al-tolerance response. 2) Functional application of Al tolerance genes for enhancing Al tolerance in crops – case studies with NRAT1, NIP1;2 and ALMT1; will be performed evaluating the levels of Al- tolerance in transgenic tomato and wheat seedlings expressing these transporters. 3) Characterization of the SbMATE interacting protein SbMBP. We will use isothermal titration calorimetry (ITC) to characterize the binding kinetics of the Al and SbMBP protein. 4) Regulation of MATE transporters via phosphorylation. We will characterize changes in the CBL/CIPK mediated changes in the transport activity of MATE transporters expressed in Xenopus oocytes via electrophysiological analysis, upon co-expression with structurally modified CIPK and CBL proteins. Identification of the phosphorylated MATE residues will be done by nanoLC-MS/MS analysis of the MATE purified protein. 5) Physiological and genetic characterization of stop1 suppressor mutants should enable the identification of new genetic and cellular components functioning in STOP1-mediated functional networks regulating Al-resistance and proton tolerance. We will perform a physiological and molecular characterization of stop1 suppressor mutants, concurrently quantifying their Al-tolerance, the magnitude of Al-induced organic acid release, and changes in gene expression of organic acid transporters involved in mediating Al-exclusion responses. The molecular identity of the suppressor mutation will be established using next-generation sequencing. 6) Analyze the genetic control and the environmental regulation of root system architecture (RSA) and the role of variation in RSA in rice and sorghum adaptation to acid soils focusing on Al toxicity and P deficiency. Using digital imagining we will quantify changes in traits defining the root architecture of rice and sorghum in response to nutrient solutions progressively modified to mimic acid soil conditions, including, but not limited to, Al-toxicity and varying phosphorus conditions. 7) Analyze differential protein expression, at the cellular level in root tips, as a function of Al exposure at acidic pH, to understand specific tissue and cell type functions. We will develop new protein labelling approaches for LC-MS/MS proteomics, thereby allowing protein quantification from various types homogeneous root cell samples harvested using laser capture microdissection (LCM). The proteomics data obtained under the various treatment will be integrated with gene expression analysis, providing information on genes that are regulated at the transcript and/or protein levels.
Progress Report
This is the final report for 8062-21000-046-000D, "Genomic and Genetic Analysis of Crop Adaptation to Soil Abiotic Stresses," which ended in March 2023. New NP301 OSQR approved project 8062-21000-049-000D, entitled "Biochemistry and Physiology of Crop Adaptation to Soil-Based Abiotic Stresses."
As aluminum (Al) readily solubilizes from clay minerals in acidic soils, its phytotoxicity significantly limits crop production. These soils comprise approximately 40% of the world's arable soils and are found in various parts of the world, including the United States and many developing countries. We have taken a multidisciplinary approach integrating five objectives to understand the physiology and molecular nature of the mechanism underlying Al resistance and tolerance to P deficiency in crop plants. Significant progress was made integrating theoretical and experimental approaches, leading to an understanding of the structural and functional relations that govern the function of membrane transport proteins mediating abiotic stress response. We identified accessory interacting proteins involved in regulating not only the expression of genes encoding for these transport proteins but also tightly binds to the transport protein, thereby modulating their transport activity. We also established the importance of the spatial distribution of the resistance responses, by demonstrating that these proteins, associated with different molecular resistance mechanisms, are differentially expressed in distinct cell types isolated from the same complex tissue, and differentially expressed throughout the root system/types and tissues. Therefore, we also investigated how plants place/distribute their root systems throughout the soil, as root distribution determines the performance under both drought and low mineral nutrient conditions. 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, thus conferring greater levels of Al tolerance and improving yields in important staple food crops.
Over the last year, significant progress has been made to Objective 1: Wheat is an experimentally recalcitrant but economically significant organism. Knowledge obtained from the model organism Arabidopsis facilitates the identification of wheat homologous genes underlying aluminum (Al) resistance. We have demonstrated that coordinated functioning between two interacting genes is required to achieve higher Al resistance levels in Arabidopsis. We have identified a couple of wheat genes that are functional homologs of the two Arabidopsis Al-resistant genes. Furthermore, the functions of the two wheat homologous genes have been confirmed in heterologous systems. The research is expected to lead to the development of molecular markers for selecting Al-resistance traits in wheat. Such knowledge will ultimately facilitate breeding programs to generate crop cultivars with enhanced Al resistance and yields on acid soils.
We made significant progress unveiling interactions between Al-resistant genes, which are critical for crop plants to achieve higher resistance levels under Al toxicity conditions (Objective 2). We have identified a candidate protein that interacts with a protein encoded by a known Al-resistant gene in sorghum. Critical experiments have been conducted to demonstrate the interactions between the two proteins. The research is expected to reveal the cellular processes underlying the resistance of sorghum plants to Al stresses and provide guidelines for the generation of crop varieties with enhanced resistance to Al toxicity and increased yields on acidic soils.
Significant progress has been made in understanding the role that the Aluminum resistance transcription factor 1 (ART1) transcription factor plays in the acid soils syndrome response. To address and dissect the role that this master regulator we have generated CRISPR edited rice lines, causing serial deletions on the ART1 gene (single, double, quadruple guides). Initial phenotyping (seed vigor and germinations rates) of the transgenic lines during the genotype has indicated that the ART1 regulatory influence spans beyond stress responses to pronton and aluminum toxicity. We have noticed a significant decrease in seed viability and germination rates in the presumably partially or non-functional ART1 lines. Although responsive to Al and proton stress, ART1 quite likely underlines the regulation of broader plant mineral nutrition / homoeostasis process. Future phenotyping of these emerging resources will provide an understanding of the role that ART1 plays in mediating Al/P and acid stress responses, as well as a molecular understanding of the functional differences among the rice ART1 allelic variation identified earlier by our group.
Significant progress has been made in Objective 5 with respect to finalizing the work associated with this objective. A dataset was deposited in the Proteomics data repository “Pride” under the accession: PXD041788. Two of the 60-month milestones are listed as partially complete as opposed to fully met due to delays caused by lingering effects of the pandemic shutdowns during which aspects of the research program got out of phase. Nevertheless, we are making up for the lost time and expect that we will have all 60-month milestones met by the end of FY2023.
Under the auspices of this work, we have continued collaboration with Tennessee State University, one of the 1890 Universities.
Aluminum artificial intelligence (Al) toxicity is an increasing problem for wheat production due to increased soil acidity caused by widely applying nitrogen-based fertilizer. Therefore, Al resistance has been an increasingly important trait for wheat breeding and yields.
Al resistance is mainly achieved by a significant homologous resistance gene in both Arabidopsis and wheat. ARS scientists at Ithaca, New York, have identified a novel Al resistance gene in Arabidopsis, which coordinately functions with the vital gene to achieve higher resistance levels. They have identified wheat sequences homologous to the Arabidopsis' second resistance gene by searching the wheat genome sequences, and functionally tested the role of the wheat homologous gene for Al resistance in Arabidopsis. It was found that the wheat genes could significantly enhance the aluminum resistance of the Arabidopsis transgenic plants. This research may result in the identification of a novel Al-resistant gene in wheat and provide guidelines for breeding wheat cultivars with enhanced Al resistance and yields on acid soils.
Accomplishments
1. Identifying interacting proteins that regulate tolerance to aluminum stress in Sorghum. Plant MATE proteins are membrane transporters that mediate the movement of organic acids that bind and immobilize toxic Al, thereby providing Al resistance. The activity of these transporters is tightly regulated by their interaction with other poorly characterized accessory proteins. Implemented biophysically characterize the molecular interaction ARS scientists at Ithaca, New York, have identified and characterized metal binding proteins (MBP) and other modifying proteins (proteins kinases which phosphorylate other proteins) which interact with the MATE transporter, thereby modifying its transport activity. These observations allowed the researchers to identify the structural regions that coordinate the physical interaction among these proteins, providing the structural and molecular information on how these proteins functionally coordinate to regulate and minimize organic acid transport thereby maintaining carbon use efficiency, while still achieving aluminum resistance. This knowledge provides an additional platform to enhancing crop yields by guiding molecular-breeding programs.
Review Publications
Cardoso, A., Gomes, F., Antonio, J., Guilherme, L., Liu, J., Li, L., Silva, M. 2022. Sulfate availability and soil selenate adsorption alleviate selenium toxicity in rice plants. Environmental and Experimental Botany. 201: Article e104971. https://doi.org/10.1016/j.envexpbot.2022.104971.
Sun, T., Zhou, X., Rao, S., Liu, J., Li, L. 2022. Protein–protein interaction techniques to investigate post-translational regulation of carotenogenesis. In: Wurtzel, E., editor. Methods in Enzymology. Amsterdam, Netherlands: Elsevier, Inc. 167:301-325. https://doi.org/10.1016/bs.mie.2022.02.001.
Koyama, H., Huang, C., Pineros, M., Yamamoto, Y. 2022. Al-induced and -activated signals in aluminium resistance. Frontiers in Plant Science. 13. Article e925541. https://doi.org/10.3389/fpls.2022.925541.
Cardoso, A., Gomes, F., Antonio, J., Guilherme, L., Liu, J., Li, L., Silva, M. 2022. Phytoene synthase: The key rate-limiting enzyme of carotenoid biosynthesis in plants. Environmental and Experimental Botany. 201:e104971. https://doi.org/10.1016/j.envexpbot.2022.104971.
Oliveira, N., Namorato, F., Rao, S., Cardoso, A., Renendeluiz, P., Guiherme, L., Liu, J., Li, L. 2023. Iron counteracts zinc-induced toxicity in soybeans. Plant Physiology and Biochemistry. 194:335-344. https://doi.org/10.1016/j.plaphy.2022.11.024.
