Location: Plant, Soil and Nutrition Research2017 Annual Report
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.
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.
During the fiscal year 2017, we have continued to make progress by meeting or exceeding the research goals in two separate but interlaced areas: 1) A significant part of the team’s research is aimed at understanding the physiological and genetic mechanisms underlying cereal crop adaptation to acid soils which comprise almost half of the world’s arable regions (including significant areas in the North East of the U.S. and developing countries). In this type of soil, aluminum ions are solubilized from clay minerals as phytotoxic free Al3+, which damages the root, generating a stunted the root systems which ultimately limits world crop productivity Understanding the basis of Al resistance mechanisms will provide a practical platform to exploit this knowledge to generate crops adapted to acid soils. 2) Research aimed at understanding the role and the genetic components governing the architecture of the root system as a means to improve plant performance and ultimately increase crop yields in marginal soils. With regards to plant aluminum tolerance research, we have extended our work identifying novel membrane transport proteins involved in mediating Al-resistance responses, as well as characterizing various accessory proteins that modulate the expression, as well as the activity of membrane transport proteins that underlie Al-resistance mechanisms: i) a novel transport protein, member of the aquaporin family, was shown to mediate the transport of the Al-malate complex from the root cell wall into the root symplasm, with subsequent Al translocation to the shoot. The activity of this transporter underlies a critical step in internal Al tolerance in Arabidopsis. ii) the novel sorghum protein SbMBP (for sorghum bicolor metal binding protein) is an aluminum (Al3+ ion) high-affinity binding protein, which binds preferentially Al3+ over other metal ions, in a pH dependent manner. SbMBP was shown to associates tightly with the SbMATE transport protein which facilitates citric acid efflux from roots into the rhizosphere where the citric acid binds and detoxifies Al3+ ions. Al3+ binding to SbMBP leads to changes in the conformation of SbMBP, resulting in disruption of the SbMATE-SbMBP interaction. Loss of this interaction results in SbMATE mediated citric acid transport out of the root, thereby activating and mediating Al-resistance. Using a series of deletion constructs of SbMBP and SbMATE, we have identified candidate interaction domains of SbMBP and SbMATE required for their interactions. We have sequenced the DNA sequences for the SbMATE and SbMBP genes from hundreds of different sorghum lines in our sorghum diversity panel. We have identified several different classes of DNA sequence variants for each gene, analyzed the correlation of specific DNA sequences with Al tolerance and root organic acid exudation. We are continuing to examine the relationship between genetic variation (differences in DNA sequence) and differences in protein function relating to sorghum Al tolerance. iii) through a comparative physiological and whole transcriptome investigation with have stablished that calcineurin B-like calcium sensors (CBLs) and CBL-interacting protein kinases (CIPKs) are involved in Al resistance responses. We have successfully identified a CBL/CIPK complex that interacts with AtMATE (an orthologue protein to SbMATE) and modulates its citrate transport activity. This protein complex constitutes part of a calcium -regulated pathway involved in the Al-resistance response, by which phosphorylation of the downstream target protein, AtMATE1, limits unnecessary carbon loss via unregulated citrate exudation, thereby regulating the abiotic stress tolerance response in a temporal and spatial manner. iv) We have continued to improve proteomic technologies to advance crop Al tolerance research. The root tip proteomes of several plant species were screened in response to control and Al toxicity treatments using multiplexed isotope coding technology. The goal of these experiments is to identify Al responsive proteins that may underlie novel Al tolerance mechanisms. The number of Al responsive proteins identified varied by species, experimental condition, and developmental stage. Previously we reported on the enhanced activity of multiple antioxidant enzymes upon Al stress in a Al tolerant sorghum line (relative to the unchanged levels observed in Al- sensitive line). The list of Al responsive proteins is being investigated further in these species to determine their biological function through literature mining and homology to proteins of known function. We have continued to refine and develop existing and new root phenotyping platforms for studying root system architecture (RSA) of crop plants. We are interested in understanding the mechanisms determining how plants place/distribute the different root types throughout the soil, as this has been shown to play a key role in improving the performance under both drought and low mineral nutrient conditions. We have developed a new phenotyping approach in which individual seedling are grown in a pouch system that allows us to maintain the root spatial characteristics. Implementation of this new platform is allowing us to increase the throughput, thereby imaging a larger number of plants in less time, a feature which is essential for genetic studies.
1. Many soils in the U.S. and parts of the world are naturally high in aluminum (Al) which can inhibit plant growth and reduce crop performance. ARS researchers in Ithaca, New York have identified novel transport proteins involved in the relocation of toxic Al within the plant, thereby unveiling a mechanism underlying genetic tolerance that can be utilized by breeders to develop crops that will better perform on high Al soils. Researchers have also identified accessory proteins and the chemical mechanism by which these systems work in two of the most important U.S. crops, corn and sorghum. This new knowledge increases our understanding of the complexity of the tolerance responses and provides a set of new targets for breeders to use for improving yields on marginal soils, thus enhancing agricultural productivity and sustainability.
2. Protein identification at the level of individual cell type to resolve plant responses to Al toxicity. Laser capture microdissection (LCM) is a microscopic technique that isolates small numbers of cells from a plant tissue for subsequent molecular or biochemical analysis. ARS scientists in Ithaca, New York, have dramatically improved a workflow originally reported in 2015 to identify proteins from cells isolated from the different regions of tomato roots. The original work suggested that distinct cell types within a specific tissue respond differently to particular biotic and abiotic challenges and that these responses must be integrated into a coordinated global response. However, the number of cells that could be reasonably isolated using the original protocol was too small to support the quantitate analysis needed to understand the biological processes involved in adaptation to growth on acid soils. The improved protocol increases the rate of cell capture by 3-4 fold making quantitative analysis using isobaric, isotope coded mass tags possible for the first time.
3. Second generation plant root imaging and data acquisition system. ARS researchers in Ithaca, New York have developed an improved design to collect digital images (2D & 3D) of the root systems of a range of agricultural crops. There is a growing need to measure both older and larger root systems to improve our understanding of the genetics that control favourable rooting traits underlying crop productivity, for future implementation in plant breeding programs. A larger imaging apparatus with a simplified control system using an innovative plant root growth media was prototyped, along with the release of new operating and imaging software. The new design presents a simplified means for collecting, managing and preserving root system images for future analysis. The new system has made data acquisition efficient by reducing imaging time, increasing system capacity, and providing safe storage of the critical experimental data.
Martin, L., Scherwood, R., Nicklay, J., Yang, Y., Muratore, S., Anderson, E., Thannhauser, T.W., Rose, J., Zhang, S. 2016. Application of wide selected-ion monitoring data-independent acquisition to identify tomato fruit proteins regulated by the CUTIN DEFICIENT2 transcription factor. Proteomics. 16:2081-2094.
Zhu, Y., Hui, L., Bhatti, S., Zhou, S., Yang, Y., Fish, T., Thannhauser, T.W. 2016. Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots. Horticulture Research. 3:16026.
Zhou, S., Okekeogbu, I., Sangireddy, S., Yi, Z., Hui, L., Bhatti, S., Hui, D., Yang, Y., Howe, K.J., Fish, T., Thannhauser, T.W. 2016. Proteome modification in tomato plants upon long-term aluminum treatment. Journal of Proteome Research. 15:1670-1684.
Ye, Z., Sangireddy, S., Okekeogbu, I., Zhou, S., Yu, C., Hui, D., Howe, K.J., Fish, T., Thannhauser, T.W. 2016. Drought-induced leaf proteome changes in switchgrass seedlings. International Journal of Molecular Sciences. 17:1251-1269.
Yuan, H., Owsiang, K., Sheeja, T., Zhou, X., Rodriguez, C., Li, Y., Welsch, R., Chayut, N., Yang, Y., Thannhauser, T.W., Pathasarathy, M.V., Xu, Q., Deng, X., Fei, Z., Schaffer, A., Katzir, N., Burger, J., Tadmor, Y., Li, L. 2014. A single amino acid substitution in an ORANGE protein promotes carotenoid overaccumulation in arabidopsis. Plant Physiology. 169(1):421-431.