Location: Plant, Soil and Nutrition Research
Project Number: 8062-21000-036-000-D
Project Type: In-House Appropriated
Start Date: Jun 28, 2013
End Date: Jun 3, 2018
Objective:
1: Determine mechanisms underlying the regulation of the major sorghum aluminum (Al) resistance gene, SbMATE, at the level of protein function, with the long term goal of identifying molecular determinants that interact with SbMATE to confer high levels of sorghum Al resistance.
1.1: Verification of SbMBP as an Al sensor and an Al-controlled switch for the SbMATE root citrate transporter.
1.2: Functional analysis of SbMBP and SbMATE proteins and their interactions.
1.3: Other protein-protein interactions modulating citrate transport mediated by SbMATE (and orthologues)
2: Conduct structure-function studies on members of a major family of cereal Al resistance proteins, the Multidrug and Toxic Compound Efflux (MATE) family of transporters, that function as root organic acid efflux transporters, to identify protein domains that play a role in conferring high levels of Al resistance.
2.1: Validation of structural and functional motifs that underlie key plant MATE transport properties.
2.2: Determination of the high-resolution structure of SbMATE by x-ray crystallography.
3: Identify and determine the roles of QTL and genes underlying these QTL identified from joint linkage/genome-wide association analysis for rice Al resistance and determine how gene-level variation influences rice Al resistance.
3.1: Fine scale map and clone the large effect rice Al resistance QTL identified on chr 12 from both bi- parental QTL mapping and GWA analysis.
3.2: Investigate the role of sequence variation for the candidate gene underlying a major QTL in the aus subpopulation, Nrat1, which encodes a rice root Al uptake transporter and determine the role this variation plays in aus Al resistance.
4: Investigate the genetic/genomic regulation of root system architecture (RSA) and the role of variation in RSA in rice and sorghum adaptation to nutrient–limited soils.
4.1: Mine the data from recently conducted joint linkage-GWA on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions.
4.2: Complete the development of a hydroponic-based system for investigating RSA in our sorghum association panel and complete GWA analysis of sorghum RSA traits in this panel.
5: Accelerate the adaptation of high throughput 3-D root imaging and image analysis to enhance the capacity of crops to adapt to climate change, increase water use efficiency, and improve nutrient use efficiency, through the genetic improvement of root architecture and physiology.
Approach:
1) Study the role of sorghum AlMBP in regulating aluminum (Al) activated citrate transport via the sorghum Al tolerance protein, SbMATE. Will use a combination of ESI-Q-TOF MS/ ion mobility spectrometry and metal-ion chromatography to determine kinetics and specificity of Al binding by AlMBP. 2) Determine if Al binding by AlMBP causes this protein to disassociate from SbMATE using in vitro pull down assays, in vivo BiFC assays, and chemical cross-linking followed by LC-MS/MS analysis. 3) Determine the functional role of the SbMBP-SbMATE interaction by expressing both proteins in heterologous systems (oocytes and yeast) to determine if this confers Al activated of citrate exudation.4) Study the role of phosphorylation in regulation of SbMATE transport function via electrophysiological analysis of citrate efflux based on co-expression of SbMATE and candidate kinase proteins (CIPKs and calcineurin B-like [CBL] proteins) in oocytes.5) Investigate the role of protein structure in transport function for the plant MATE proteins that mediate citrate efflux and are involved in Al tolerance. Will determine the 3D crystal structure of SbMATE and use this structural model to direct functional analysis of SbMATE transport in oocytes. 6) After identifying altered SbMATE-type transporters that show enhanced function, the effects of these variants in plants will be determined by expressing SbMATE variants in transgenic Arabidopsis seedlings, and determining changes in Al tolerance. 7) In studies on rice Al tolerance, we will mine genome-wide association (GWA) data to identify/test candidate rice Al tolerance genes by a combination of high resolution mapping, molecular analysis in rice, expression of candidate Al tolerance genes in transgenic rice, and functional analysis of candidate transporter genes such as the Nrat1 Al transporter in heterologous systems (oocytes and yeast). 8) For research on root system architecture, we will mine data from joint linkage-GWA analysis on rice RSA traits to identify regions of the rice genome controlling root traits that play a role in nutrient acquisition (P, water and N) under limiting conditions. This will involve a combination of fine scale mapping, mRNA seq analysis of candidate genes, expression of candidate RSA trait genes in transgenic rice, and the verification of functionality of different root architectures by looking at performance in soil under limiting (low water, N or P) conditions.
