Location: Plant, Soil and Nutrition Research
2023 Annual Report
Objectives
OBJECTIVE 1: Determine how aluminum and heavy metal stress tolerance, organic acid homeostasis, and phosphorus allocation are balanced in crop plants (Sorghum, maize, and rice) and Arabidopsis by aquaporin (AQP), SULTr, and MATE (multi-drug and toxic compound extrusion) transporters.
Sub-Objective 1.A: Functional studies and characterization of the roles of AQP members in heavy metal and aluminum resistance.
Sub-Objective 1.B. Determining protein domains associated with the transport mechanics and substrate specificity of SbMATE.
Sub-Objective 1.C. Establishing the functional roles and properties of the members of the OsSULTR3 clade.
OBJECTIVE 2: Determine the physiological signaling pathway(s) that modulate STOP1 activity, the master transcription factor regulating plants’ adaptation to low pH, aluminum stress, and low phosphate availability.
Sub-Objective 2.A. Identify new genetic and cellular components critical for STOP1-mediated resistance to low-pH and Al stresses.
Sub-Objective 2.B. Studies on the interactions between STOP1, phytohormones, and low-pH stress and their roles in achieving resistance to low-pH stresses in plants.
OBJECTIVE 3: Identify and functionally characterize signaling networks controlling the expression of and/or the activity of membrane proteins mediating the transport of organic acids (e.g., malate and citrate) and phosphate, underlying aluminum chelation/detoxification and phosphate allocation processes.
OBJECTIVE 4: Develop new methods to spatially resolve and quantify protein abundance in just a few root cells to improve functional analyses of proteomes in response to abiotic stress.
Approach
1) Identify the role of members of the SULTr, MATE, and Aquaporin family of plant membrane transporters as the basis of key agronomic traits. We will study their functional characteristics (e.g., efflux/influx of substrate fluxes and electrophysiological fingerprints by expressing them in heterologous systems (e.g., oocytes and yeast). Changes in functionality upon structural changes will highlight structural motifs underlying the protein’s functional properties/signature. Phenotypic analysis of single and double loss of function mutants in transgenic Arabidopsis seedlings will establish the genetic relations and interactions among the different transport families in response to abiotic stress (e.g., Al tolerance, heavy-metal and P transport.
2) Physiological and genetic characterization of stop1 suppressor mutants will enable the identification of genetic and cellular components functioning in STOP1 (a master transcription factor regulator in the acid soil syndrome response) -mediated functional networks regulating Al and proton tolerance. We will correlate root organic acid release and changes in the gene expression of organic acid transporters in response to metal-toxicity stress. We will use next-generation sequencing (NGS)-based mapping-by-sequencing approaches to identify and confirm the molecular identity of the suppressor mutation and characterize their cellular and molecular functions by examining the tissue specificity of the gene expression, the subcellular localization, and putative functional roles.
3) To dissect the network pathways controlling the expression of and/or the activity of ALMT and MATE membrane proteins, we will structurally modify both interacting and transport proteins. We will examine changes in their localization/endomembrane trafficking pathways tagging them with fluorescent reporters (e.g., GFP or YFP) and transiently expressing in tobacco and xenopus oocytes. We will probe for changes in the protein-protein interactions (e.g., CBL/CIPK complex interacting with, phosphorylating, and modulating the MATE transporter activity) by measuring changes in fluorescent complementation (BIFC) relative to those observed for the native proteins. Changes in protein-protein interactions will be correlated with potential changes in the overall electrogenic activity of the transporter assayed using a two-electrode voltage clamp.
4) The development of new methods to spatially resolve and quantify protein abundance in just a few root cells to improve functional analyses of proteomes in response to abiotic stress. Samples (˜ 100 cells) will be harvested using LASER Capture Microdissection (LCM) will be digested, extracted in one single step/vessel, to perform proteomic studies with a protein-label-free LC-MS/MS-based protocol. The proteomics data obtained under the various stress treatments will be integrated with gene expression analysis, thereby providing information on genes regulated at the transcript and/or protein levels.
Progress Report
This is a new project that recently started on April 2023. For more details, please see the final annual report for 8062-21000-046-000D.
Since April, we have made limited progress on some of the first-year milestones of the new project. Efforts were placed on the design and construction of numerous DNA plasmids and mutant-lines isolation, thereby generating the resources for future milestones of the first three objectives. These include: a) single and double Arabidopsis and Rice mutants for studies related to the functional characterization of aquaporin in regard to heavy metal and aluminum resistance; b) construction of transporter chimeras multi-drug and toxin compounds extrusion (MATES) and sulfate transporter (SULTrs) to identify structural motifs associated with functional characteristics, and to examine protein turnover/stability and protein-protein interactions between these and other accessory proteins.
For Objective 4, optimization of the preparation of plant root material is underway as is the recommissioning of the P/ACE MDQ CE system and a request has been made to fund the acquisition of a new c-isoelectric focusing electrophoresis (IEF) system to replace the P/ACE MDQ system.
Under the auspices of this work, we have continued collaboration with Tennessee State University, one of the 1890 Universities.
Accomplishments
