Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Microbiology & Cell Biology
Non Technical Summary
Ensuring plant survival during environmental stress is necessary to feed our growing human population particularly in the face of fluctuating precipitation and temperature regimes linked to climate change. The release of carbon (C)-rich root exudates is a key mechanism by which plants manage the root microbiome to withstand environmental stress, resulting in a "C-cost" diverted from growth and metabolism. The proposed research project will determine how plants and soil microbes adapted to different climate regimes balance the cost-benefit of maintaining the root microbiome under drought and heat stress. I will use a greenhouse experiment to impose four environmental stress treatments on the grass species blue grama: ambient, heat, drought, heat+drought in soils collected across a natural precipitation gradient. I will measure plant C allocation to growth and root exudates using 13CO2-labelling and plant physiology and determine how stress-induced shifts in C allocation alter the active rhizosphere using metagenomics (stable isotope probing, SIP). Deliverables will include one peer-reviewed, first-author publication contributed to my dissertation materials, and three co-authorships. This proposal directly addresses the AFRI Priority Area of Plant Health and Production and Plant Products as it will result in new knowledge regarding the adaptation of plants and microbes to environmental stresses and the consequences of altered plant C allocation for agricultural landscapes. This Predoctoral Fellowship is relevant to the AFRI EWD goal of Advancing Science because it will allow me to pursue my career goal of improving agricultural sustainability by developing climate-smart agricultural practices prioritizing healthy plants and soils.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The main objective of this proposal is to determine the effect of precipitation legacy on C allocation and microbiome restructuring of blue grama under environmental stress.While my training has prepared me to design and conduct fundamental research in microbialecology, I am most inspired to bridge interactions of the microbial world from soil to plants. I amcurrently transitioning to a NIFA-funded project that focuses directly on the mechanisms thatunderlie above- and belowground interactions between plants and microbes in systemsexperiencing climate stresses such as heat and drought. The goals of that project are focused onunderstanding plant allocation of C belowground under heat and drought stress, the effects ofthose stresses on the soil microbiome, and how plant and microbiome responses are linked.Building on this, here I am proposing to expand our current study to include a range of sitesacross Montana that represent a strong precipitation gradient. By incorporating a broader naturalgradient, I will drastically broaden the scope and robustness of this research to improvepredictability across ecosystems. The interdisciplinary nature of the project, which includes myprimary advisor, Dr. Brent Peyton (environmental bioengineer), and collaborators Dr. RebeccaMueller (microbial ecologist) and Dr. Danielle Ulrich (plant physiologist) will increase myprofessional skillset. In carrying out this project I will achieve my goals of gaining experience in plant physiology andmicrobial -omics approaches and I will learn new laboratory techniques and analyses that willsignificantly benefit my competitiveness as a future career applicant in agricultural sciences.
Project Methods
Approach. Experimental Design. I will collect soil from four additional locations in Montana where blue grama is naturally found representing a 400 mm precipitation gradient. Blue grama seeds will be purchased from a reputable seed dealership (Wind River Seed, WY) and grown in a mix of 10% field-collected soil in pasteurized potting mix. A 10% inoculum is sufficient to allow proliferation of native soil microbiomes while minimizing soil nutrient and texture differences. To ensure establishment, the plants will be allowed to grow for 30 days prior to application of environmental stress treatments.Sample collection. After 30 days, plants will be randomly assigned to one of four treatment conditions for 60 days: Ambient, drought, heat, or heat + drought, n=5 for each. Drought treatments will reflect 50% precipitation reductions from the ambient used to maintain robust plant growth and heat treatments will reflect 3 ?C above ambient. At each time point (20, 40, 60 days), 5L of 99% 13CO2 (Sigma-Aldrich) will be injected into sealed growth chambers for several hours to ensure full uptake by the plants. After 72 hr, root exudate, shoot, and root samples will be collected and analyzed with an isotope ratio mass spectrometer at the Oregon State University Stable Isotope Laboratory. The amount of 13C label allocated to roots and root exudates will be calculated as the 13C atom percent excess as in Karst et al. (2017) to determine the root exudate:root mass ratio. This ratio can be scaled up to estimate root exudation at field scale, informing ecosystem C fluxes. Plant physiology including root and shoot biomass, photosynthesis and respiration will be measured at each time point. Soil samples will be collected by shaking soil off each plant which will then be flash-frozen and transferred to -80 ?C for long-term storage. For plant physiology the total sample count will be 310 (5 sites, 4 treatments, 3 time points, 5 replicates, and ten pretreatment samples). Soil chemistry data including pH, TOC, and total nitrogen and phosphorous will be collected for each of the soil sites. Due to the cost of metagenomics, I will focus microbial sampling on the low-, mid-, and high- precipitation history sites (n=3), and only the ambient, drought, and heat+drought treatments at the final timepoint. I will use soils without plants (no-plant controls) to identify the contribution of C-fixing bacteria relative to plant photosynthates. This brings the total metagenome count to 27 (3 sites, 3 treatments, 1 timepoint with paired no-plant controls, plus 9 background samples (n=3 for each site).Microbiome sequencing. For metagenomics, DNA will be extracted from 2 g of soil, and heavy DNA (13C) will be separated using cesium-chloride gradients as described by Buckley et al.to identify the microbial community actively taking up newly fixed C. Metagenomic library construction and sequencing across one lane of the Illumina NovaSeq6000 will be conducted at the University of Oregon Genomics and Cell Characterization Core Facility. Metagenome-assembled genomes (MAGs) for Bacteria and Archaea will be constructed. High quality MAGs will be classified taxonomically using the Genome Taxonomy Database and functionally annotated using the program RASTand Prokka. As reconstruction genomes of eukaryotic microbes from environmental samples is generally unsuccessful due to the complexity of mixed communities in diverse soil we will classify unassembled sequences against known databases (e.g., community aggregated traits), including Carbohydrate Active Enzymes (CAZyme), Protein Families (PFAM)and UniProt, to gain insights into the functional potential of eukaryotes present across the research sites, with a particular focus on fungi present in soils. All statistical analyses will be performed in R using R Markdown to create runnable, annotated scripts for enhanced reproducibility.Feasibility. Although isotope labeling can greatly advance our understanding of plant C flows to root exudates, pulse labeling does not deliver reliable C balances. However, complementing isotope labeling with additional C allocation measurements including respiration, photosynthesis and root exudate quantitation provides a reliable overall C budget. In addition, the high diversity of microbial communities in soils complicates metagenomic assemblies, but our approach of acquiring higher coverages (n=3) in background soils will improve confidence in our assemblies.The work described above will provide new knowledge of plant-microbe interactions under heat and drought stress that will be used in outreach efforts, by co-mentor Dr. Ulrich in teaching materials, and published in peer-reviewed articles. Evaluation milestones will include peer-reviewed publications and feedback from outreach presentations.