Location: Plant, Soil and Nutrition Research
2020 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
Wheat is an experimentally recalcitrant but economically important organism. Knowledge obtained from the model organism, Arabidopsis, could facilitate the identification of wheat homologous genes responsible for aluminum (Al) resistance. We have demonstrated that coordinated functioning between two interacting genes is required for achieving higher levels of Al resistance in Arabidopsis. We have identified wheat genes that are functional homologs of the two Arabidopsis Al resistance genes. The research is expected to develop molecular markers for selection of Al-resistance traits in wheat. Delivery of such knowledge will ultimately facilitate breeding programs aimed at generating crop cultivars with enhanced Al resistance and yield on acid soils.
Interactions between Al resistant genes are critical for crop plants to achieve higher levels of resistance under Al toxicity conditions. We have identified a candidate protein that interacts with a transport protein that mediates Al resistant responses. Critical experiments have been conducted to demonstrate that these proteins physically interact with transporters to modulate transport activity. The research is expected to reveal the cellular processes underlying the resistance of sorghum plants to Al stresses and to provide guidelines for generation of crop varieties with enhanced resistance to Al toxicity and increased yield on acidic soils.
While we have demonstrated the efficacy of the protein-level labeling strategy using both thiol and amine targeted reagents with respect to their abilities to generate improved quantitative data through reduced problems associated with co-elution of near-isobaric labeled peptides, the proteomic coverage of these experiments was less than hoped for. Fortunately, several developments have become available commercially [MS3 quantification and Real Time Search (RTS) capabilities] that obviate the need for protein level labeling. We had experimented with MS3 quantification earlier and were aware of its power to reduce the interference of near-isobaric labeled ions. However, the need to carry out a time consuming MS3 analysis on all precursor ions detected (many of which produce unidentifiable spectra or are otherwise unproductive) reduced the number of identified/quantified peptides, which in turn reduced the coverage of the proteome to unacceptable levels. The coupling of these two developments (RTS-MS3) makes it possible to identify the quantifiable precursor ions at the MS2 stage such that the time consuming MS3 analysis is only carried out on productive ions. This saves significant amounts of time and allows us to focus our analysis on the quantifiable ions, resulting in higher proteome coverage and accurate quantitative data. Our preliminary experiments utilizing the RTS-MS3 approach have been excellent and it has now become our standard operating procedure.
We continue to develop and refine our protocols in tomato and have published a comparative study of protein expression in the Al-sensitive root transitional zone focusing on epidermal and cortical cells collected by laser capture microdissection. This paper describes an efficient laser capture microdissection-tandem mass tag-quantitative proteomics analysis platform for the study of Al sensitive root cells. The analytical procedure has a broad application for proteomics analysis of spatially resolved cells from complex tissues. This study has provided comprehensive proteomics datasets of epidermal and outer-cortical cells of the root-tip transition zone of Al-treated tomato seedlings. The proteomes from the Al-sensitive root cells are valuable resources for understanding and improving Al tolerance in plants. Data sets have been deposited in the Proteome change Data Repository.
Under the auspices of this work, one of our team scientists has been able to continue his collaboration with a professor at Tennessee State University, one of the 1890 Universities. Through this effort he has been able to fulfill our obligation to support the 1890 Universities and the under-represented minority groups they serve.
Accomplishments
1. Improving aluminum resistance in wheat. Aluminum (Al) toxicity is an increasing problem for wheat production due to elevated soil acidity caused by the application of nitrogen-based fertilizer. Therefore, Al resistance has been an increasingly important trait for wheat breeding and production. Researchers in Ithaca, New York, identified a novel Al resistance gene in Arabidopsis, which coordinately functions with previously identified genes to achieve higher levels of resistance. The function of this gene in Al resistance was accomplished in yeast. Identification of novel Al resistance gene in wheat was achieved and breeding wheat cultivars with enhanced Al resistance and yields on acid soils.
2. Protein differences among cell types influence crop stress tolerance. The physicochemical properties of acid soils give rise to the acid soil syndrome, characterized by aluminum (Al), proton and manganese toxicity; calcium, molybdenum, magnesium, and phosphorus deficiencies; legume nodulation failure; and increased susceptibility to plant disease, which all negatively affect plant productivity. To address these issues, scientists at Ithaca, New York, working with colleagues at Tennessee State University, have discovered an efficient LASER capture method to test Al sensitive root cell types. From the ability to study the Al-sensitive root, cell types reported are valuable resources for understanding and improving Al tolerance in plants.
3. A single plant transport protein can improve different take-up sugars in apples. Improvement of apple fruit quality and tree fertility has been one of the major goals in apple breeding programs. An incomplete understanding of the genetics and fertilization of apples make genetic improvement for apple breeders. ARS scientists at Ithaca, New York, working with colleagues at Cornell University, used a multidisciplinary approach to characterized an apple sugar transport protein (STP13) in pollen grains. This protein can mediate the uptake of both sucrose and hexose, essential for the pollen tube growth. Sorbitol, a major product of photosynthesis and transport carbohydrate in apple, modulates pollen tube growth via known transcription factors (MYBs). The mechanistic findings in this work should assist in selection in apple breeding programs targeting fertility traits.
Review Publications
Magalhaes, J., Pineros, M., Maciel, L., Kochian, L. 2018. Emerging pleiotropic mechanisms underlying aluminum resistance and phosphorus acquisition on acidic soils. Frontiers in Plant Science. 9:1420.
Riedelsberger, J., Vergara-Jaque, A., Pineros, M., Dreyer, I., Gonzalez, W. 2019. Extracellular cation binding pocket is essential for ion conduction of OsHKT2;2. Biomed Central (BMC) Plant Biology. 19(1);316.
Huber, A., Melcher, P., Pineros, M., Setter, T., Bauerle, T. 2019. Signal coordination prior to, during, and after stomatal closure in response to drought stress. New Phytologist. 224:675-688.
Li, C., Meng, D., Pineros, M., Mao, Y., Dandekar, A.M., Cheng, L. 2020. A sugar transporter takes up both hexose and sucrose for sorbitol-modulated in vitro pollen tube growth in apple. The Plant Cell. 32:449-469. https://doi.org/10.1105/tpc.19.00638.
Maity, K., Heumann, J., Mcgrath, A., Kopcho, N., Hsu, P., Lee, C., Mapes, J., Garza, D., Krishnan, S., Morgan, G., Hendargo, K., Klose, T., Rees, S., Medrano-Soto, A., Saier, M., Pineros, M., Komives, E., Schroeder, J., Chang, G., Stowell, M. 2019. Cryo-EM structure of OSCA1.2 from Oryza sativa: Mechanical basis of potential membrane hyperosmolality-gating. Proceedings of the National Academy of Sciences. 116(28):14309-14318.
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). https://doi.org/10.3390/proteomes6020015.
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). https://doi.org/10.1080/15592324.2017.1422469.
Zhao, Z., Gao, X., Ke, Y., Chang, M., Xie, L., Li, X., Gu, M., Tang, X., Liu, J. 2019. A unique aluminum resistance mechanism conferred by aluminum and salicylic-acid-activated root efflux of benzoxazinoids in maize. Plant and Soil. 437:273-289. https://doi.org/10.1007/s11104-019-03971-9.
