Location: Plant, Soil and Nutrition Research
2019 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
Researchers have made significant progress in the development and integration of various theoretical and experimental approaches to gain insight into the relationship between the structure and the function of membrane transport proteins mediating abiotic stress response. In the theoretical aspects researchers have initiated the construction and refinement of protein models based on structural homology to know crystal structures; in combination with future molecular dynamics, this will allow one to understand the structural –conformational changes that take place during the transport process. Researchers have also made progress in the experimental front: 1) Researchers have continued the functional characterization of these transporters by monitoring the activity of the proteins as expressed in the membrane of heterologous systems by performing the characterization of the purified and artificially reconstituted protein. Researchers have also developed alternative and novel methodologies that have allowed us to express membrane transporters that fail to express using conventional protocols, by expressing them in a soluble fraction. This research and methodology advancements are allowing researchers to move forward in the structure-function analysis of various families of transporters and establish the molecular nature of the transport processes.
Objective 2: Researchers have identified and characterized novel accessory proteins which Interact with Al resistant genes encoding membrane transport proteins, thereby modulating their efficiency, resulting in higher levels of resistance under Al toxicity conditions. Using a variety of biochemical and biophysical approaches researchers have successfully demonstrated the physical interaction, and specificity in the formation of these accessory-transporter complexes. The interaction of these accessory proteins with the membrane transport protein, results in their transport activation or repression, thereby modulating their activity. Being at the core of the physiological process underlying stress responses, this research is expected to reveal the cellular processes underlying the resistance of sorghum and other crop species to Al stresses, consequently providing guidelines for the generation of crop varieties with enhanced resistance to Al toxicity and increased yields on acidic soils.
Objective 3: A master transcription factor that controls the resistance Arabidopsis plants to low pH and Al stresses has been identified previously. However, it remains unknown for how this transcription factor regulates the unknown cellular components to achieve the tasks. With a genetic approach, researchers have identified several key loci that are involved in the above-mentioned transcription factor-mediated processes leading to resistance to low pH and Al stresses in Arabidopsis. A couple of candidate genes have been identified. The results are expected to reveal the cellular networks and signal transduction pathways underlying plants resistance to Al toxicity.
Objective 4: Researchers have implemented in-house imaging platforms to examine the plasticity of root architecture traits in response to varying phosphate (P) environments. By quantifying various root traits in sorghum plants grown in different P regimens, researchers have established a dependency of several of the root trait(s) previously associated with P-efficiency have a strong dependency on the environmental conditions (i.e., phosphate levels).
Objective 5: Researchers have demonstrated the efficacy of the protein-level cysteine labeling strategy as a means to decreasing the impact of ratio distortion caused by co-isolation of near isobaric labeled precursor ions. This has been demonstrated in the test case of heat stress in the floral tissue of tomato. A number of proteins (~100) were identified whose abundance ratio changed significantly between the control and heat treated samples. Among them were many proteins known to be affected by heat stress that serve to validate our approach. Others not known to be connected to heat stress tolerance become objects for further work and perhaps future targets for those trying to breed heat resistant cultivars. In the process, we also demonstrated the utility of electrophoresis as a means of protein-level pre-fractionation of multiplexed, labeled protein samples. A manuscript has been prepared (365284) and submitted to a peer reviewed journal and is currently being revised. Data sets have been deposited in the ProteomeXchange date repository.
The background sample-analytical sample approach that was incorporated in our original experimental design has been replaced by a “two proteome” interference model. This new strategy has been adopted because it is more efficient and has been demonstrated to be effective in the literature.
Researchers continue to develop and refine their protocols in tomato and have completed a comparative study of protein expression in the Al-sensitive root transitional zone focusing on epidermal and cortical cells collected by laser capture microdissection. These samples contained 20-25 ug of protein from ~ 90,000 cells and resulted in the quantification of 3879 proteins. To our knowledge, this is the most comprehensive proteomic data set collected concerning on individual cell types from the Al-sensitive root transition zone. A manuscript has been prepared (365306, see below) and submitted to a peer reviewed journal and is currently in revision. Data sets have been deposited in the ProteomeXchange Data Repository.
New laser capture microdissection protocols had to be developed due to acquisition of a new instrument. Overall, this is a positive development but does involve re-optimization of existing protocols because the operating principles of the two LCM instruments are significantly different. This work is ongoing and is making steady progress. An ancillary development that has been achieved is a protocol for remote operation of the LCM instrument that allows collaborators at Tennessee State University to access this instrument remotely. This saves them the time and trouble of traveling to a USDA-ARS location in Ithaca to use the instrument and leads to a more efficient allocation of this research resource.
Under the auspices of this work, an ARS scientist in Ithaca, New York, has been able to continue collaboration with Tennessee State University, one of the 1890 Universities. This effort helped to fulfill an obligation to support the 1890 Universities and the under-represented minority groups they serve.
Accomplishments
1. Gene in wheat that yields more in acid soils. Environmental factors such as soil acidity, low rainfall, extreme temperatures, salinity, among others, contribute to drought that affects agricultural productivity and food security. Wheat is an experimentally recalcitrant but economically important organism. Knowledge obtained from the model organism Arabidopsis could accelerate the process of the identification of homologous genes responsible for resistance against Al toxicity in wheat. ARS researchers in Ithaca, New York, have identified a gene in wheat, which is a functional homolog of an aluminum (Al) resistant gene in Arabidopsis. We have demonstrated that as in Arabidopsis, the protein encoded by this wheat gene is involved in contributing resistance to Al stress. This research will assist in the development of molecular markers for Al-resistant genes from economically important crops. Delivery of such knowledge will ultimately facilitate breeding programs aimed at generating crop cultivars with enhanced Al resistance and yields on acid soils.
2. Provide guidelines for crop varieties with enhanced resistance to Al toxicity. The interactions between Al resistant genes are critical for crop plants to achieve higher levels of resistance under Al toxicity conditions. ARS researchers in Ithaca, New York, have identified candidate proteins that physically interact and modulate the activity of protein encoded by known Al resistant gene in sorghum. This 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 yields on acidic soils.
3. Understanding of the structure of proteins involved in mediating stress responses in a crop species. Using a multidisciplinary approach by combining biochemical, biophysical and computational studies, an inferred structural model for a protein from rice that is involved in recognizing and decoding drought-related stress signals was recognized. This structural model explains the mechanisms by which the protein senses physical forces, such as those experienced during drought, and translates them into biological (biochemical) signals, thereby evoking a variety of physiological and molecular responses. This research provides an understanding of the structure of proteins involved in mediating stress responses in a crop species, and provides the basis to understand how plants respond and adapt in response to changes in the environment.
Review Publications
Arbelaez, J., Maron, L., Jobe, T., Pineros, M., Rebelo, A., Famoso, A., Ma, Q., Fei, Z., Kochian, L., McCouch, S. 2017. Aluminum resistance transcription factor 1 (ART1) contributes to natural variation in rice aluminum resistance. Plant and Soil. 1(4):1-19. https://doi.org/10.1002/pld3.14.
Kykurugya, M.M., Mendonca, C.M., Solhtalab, M., Wilkines, R.A., Thannhauser, T.W., Aristilde, L. 2019. Multi-omics analysis unravels a segregated metabolic flux network that tunes co-utilization of sugar and aromatic carbons in Pseudomonas putida. International Journal of Polymer Science. 1-16. https://doi.org/10.1074/jbc.RA119.007885.
