Location: Plant, Soil and Nutrition Research
Project Number: 8062-21000-046-000-D
Project Type: In-House Appropriated
Start Date: Jun 4, 2018
End Date: Mar 14, 2023
Objective:
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.
