Research Project: Genetic and Genomic Characterization of Crop Resistance to Soil-based Abiotic Stresses

Location: Plant, Soil and Nutrition Research

2021 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
Significant progress has been made to Objective 1. Aluminum (Al) resistance processes in many crop species involve coordinating various transport processes mediated by membrane proteins from various families. We have identified potential structural protein domains which define the transporter's function in some of the members of these families. A comprehensive understanding of the molecular basis of their functionality, which defines their role in planta, will provide a platform to identify the most effective membrane transport alleles in conferring Al- resistance. Wheat and sorghum are experimentally recalcitrant but economically significant species. We have exploited the knowledge obtained from aluminum (Al) resistance processes occurring in other model organisms, such as Arabidopsis, to facilitate the identification and discovery of homologous genes in wheat and sorghum. We have demonstrated that attaining higher Al resistance levels in Arabidopsis require the coordination of two interacting genes. We have identified a couple of wheat genes homologs of these two Arabidopsis Al-resistant genes, and their functions 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. Significant progress has been made to Objective 2. Interactions between Al-resistant genes are critical for crop plants to achieve higher resistance levels under Al toxicity conditions. We have identified candidate proteins that interact and modulate a membrane transport protein encoded by a known Al resistant gene in sorghum. Experiments have been conducted to demonstrate the interactions between the two proteins and unveil the molecular process's nature mediating the transport process's regulation. The research is expected to reveal the interrelated cellular processes that underlie the resistance of sorghum plants to Al stresses and provide guidelines for the generation of crop varieties with adequate physiological responses to cope with Al toxicity in acidic soils. Slow progress has been made with respect to Objective 5 due to the impact of COVID 19 restrictions. We continue to develop and refine our protocols concerning the implementation of real-time-time search MS3 (RTS-MS3) quantification and its applications to quantitative proteomics and have published a comparative study of RTS-MS3 verses both synchronous precursor selection MS3 (SPS-MS3) and MS2 on two different tribrid mass spectrometry systems. This paper demonstrates that RTS-MS3 offers the most accurate quantitative information and the greatest depth of proteome coverage of the tested approaches. The new strategy enabled the detection of a novel metabolic pathway that contributes to H2S-induced cell apoptosis. The results of this study justify our adoption of this approach (as opposed to protein level labeling as originally proposed) and alteration of our experimental design. The recommended analytical procedure has a broad application for quantitative proteomics analysis, including spatially resolved cells from complex tissues. Data sets have been deposited in the ProteomeXchange Data Repository. Under the auspices of this work, we have continued collaboration with Tennessee State University, one of the 1890 Universities. We have fulfilled our obligation to support the 1890 Universities and the under-represented minority groups they serve through this effort.

Accomplishments
1. The broad use of nitrogen-based fertilizers and continuous harvesting of high-yielding crops results in increasing soil acidification. The persistent toxicity experienced by plants to some soil-based metals under these soil conditions results in overall growth inhibition and a severe reduction in yields. Scientists at ARS Ithaca, New York, have identified genes that encode proteins that enable crop plants to overcome aluminum toxicity in acid soils by transporting aluminum-binding compounds out of the vulnerable growing plant regions. Identifying how the various proteins transport and regulated aluminum at the cellular and whole plant level enhance aluminum detoxification and resistance response. The finding in this work is a guide to breeding programs aimed at enhancing the productivity of staple crops grown in marginal acid soils.

Review Publications
Karavolias, N.G., Greenberg, A.J., Barrero, L.S., Maron, L.G., Shi, Y., Monteverde, E., Pineros, M., Mccouch, S. 2020. Low additive genetic variation in a trait under selection in domesticated rice. Genes, Genomes, Genetics. 10(7):2435-2443. https://doi.org/10.1534/g3.120.401194.
Li, C., Dougherty, L., Coluccio, A., Meng, D., El-Sharkwy, I., Borejsza-Wysocka, E., Liang, D., Pineros, M., Xu, K., Cheng, L. 2020. Apple ALMT9 requires a conserved C-terminal domain for malate transport underlying fruit acidity. Plant Physiology. 182(2):992-1006. https://doi.org/10.1104/pp.19.01300.
Wang, Y., Yu, W., Yu, C., Cai, Y., Lyi, S.M., Tang, X., Dong, D., Kang, Y., Liu, J. 2020. An exclusion mechanism is epistatic to an internal detoxification mechanism in aluminum resistance in Arabidopsis. Biomed Central (BMC) Plant Biology. 20:122. https://doi.org/10.1186/s12870-020-02338-y.