Source: UTAH STATE UNIVERSITY submitted to
NANOPARTICLES PRIME CROP DEFENSES FOR ABIOTIC STRESS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1012401
Grant No.
2017-67021-26603
Project No.
UTA-01372
Proposal No.
2016-08771
Multistate No.
(N/A)
Program Code
A1511
Project Start Date
Jun 1, 2017
Project End Date
May 31, 2020
Grant Year
2017
Project Director
Britt, D.
Recipient Organization
UTAH STATE UNIVERSITY
(N/A)
LOGAN,UT 84322
Performing Department
Biological Engineering
Non Technical Summary
Drought is a major reoccurring global abiotic stress that negatively impacts crop quality and quantity. Urbanization, and domestic water use place further pressure on agriculture by competition for arable land and water. Improving crop resilience to abiotic stressors, such as drought, is a cornerstone for maintaining our agricultural productivity. The goal of this research is to improve drought tolerance in plants by boosting inherent defense mechanisms through exposure to engineered nanoparticles containing copper, zinc, and silicon. These metals are plant micronutrients present in fertilizers, but not specifically as nanoformulations, which exhibit unique properties and activity due to their size and geometry. The interactions of the nanoparticles with root-colonizing microbes that improve plant response to drought will also be investigated, with the aim of utilizing the plant's beneficial microbiome to maximize drought protection. Wheat will be used in this research as it is one of the three major grains globally consumed that is often grown in drought-susceptible regions. The resilience to drought will be characterized as a function of nanoparticle formulations and concentrations under controlled greenhouse conditions. Root and leaf morphology (size, mass, thickness), photosynthetic activity, and leaf water content will be measured. The colonization potential of the beneficial bacteria will be studied to understand the importance of forming protective biofilm structures on the plant root surface. Assessment of the regulation of plant genes associated with drought protection will aid in the optimization of nanoparticle formulations. Nanoparticle surface coatings will be developed to improve particle availability to the plant, allowing for safe and effective use in agriculture to prime crops against abiotic stress such as drought.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2031549202034%
2031549200033%
2031549106033%
Goals / Objectives
Drought is a critical abiotic stress intensified by climate change that challenges crop production and quality. The proposed research will investigate nanoparticle (NP) applications to soils with the overarching aim to improve agricultural output under drought stress by priming plant defenses. Plants have evolved varied systems of drought tolerance that encompass morphological and physiological changes along with altered production of protective proteins and metabolites. A focus on drought-priming effects of bare and capped CuO, ZnO and SiO2 NPs is explored here, paralleling advancements in NPs as fertilizers and antimicrobials.Although the plant alone mounts effective mechanisms to alleviate drought stress, in nature plants exist in association with an array of microbes that colonize the plant both internally and on external surfaces. Specific microbes associated with plant roots along with select micronutrients, combine to trigger enhanced drought stress protection. For this research, NP-microbe-plant interactions in the rhizosphere will be considered as a possible mode of NP-induced priming of plants against drought. Wheat and its associated microbiome, including a drought-protectant isolate, will be studied in three soil types and sand with and without water stress to reveal molecular to morphological mechanisms by which NPs protect plants against drought.The long-term goal of the proposed research is to improve plant response to drought by elucidating the mechanisms of NP-plant-microbe interactions that prime the plant against abiotic stress. Our hypothesis is that NPs exhibit a priming effect against abiotic stress of drought that is dependent on NP composition and concentration.The primary objectives of this research are to:• Objective-1. Identify NP-dose relationships that prime plants for drought protection• Objective-2. Optimize NP activity / bioavailability through NP coatings• Objective-3. Identify the protectant pathways in the plant and rhizosphere primed by the NPs.Building on our extensive research on metal oxide NPs with plants and microbes, this research will generate NP-dose relationships that prime plants against imposed drought stress in wheat grown in several soil types. The resilience to drought will be characterized as a function of nanoparticle formulations and concentrations under controlled greenhouse conditions. Root and leaf morphology (size, mass, thickness), photosynthetic activity, and leaf water content will be measured. The colonization potential of the beneficial bacteria will be studied to understand the importance of forming protective biofilm structures on the plant root surface. Assessment of the regulation of plant genes associated with drought protection will aid in the optimization of nanoparticle formulations. Nanoparticle surface coatings will be developed to improve particle availability to the plant, allowing for safe and effective use in agriculture to prime crops against abiotic stress such as drought.
Project Methods
In Objective-1 the effective doses of CuO, ZnO and SiO2 NP or mixtures of NPs at conferring drought protection on the wheat plants grown in three soils will be determined based on measurements of shoot height, stomatal conductance, chlorophyll fluorescence, root morphology and osmolytes. Objective-2 will repeat the experiments and analyses in Objective-1 using PEG-capped and glycine betaine-capped, and proline-capped CuO, ZnO and SiO2 capped NPs and mixtures. The most effective doses determined in Objectives 1 and 2 will be compared statistically. Only the most effective doses (capped and/or uncapped) of the CuO, ZnO and SiO2 NPs or mixtures will be used in Objective-3 experiments. Results from Objective 3 will reveal the underlying biochemistry, molecular basis, and mechanisms for the responses observed in NP-primed plant-microbe systems displaying protection against drought stress. The metabolites and cell signaling plant growth regulators are those that we predict to be involved in drought protection. Thus, we will feed our results into established pathways. This work provides a platform from which can be extended to other disciplines. We view NPs, even those that release ions, as point sources or reservoirs of metals that may exert the priming effect that is amplified through NP properties.Through these experiments we will generate data that describe NP effects on gene expression and metabolic and morphologic response of the plant, and evolution and solubility of NPs as influenced by rhizosphere processes that in turn affect the plant response. Exploratory statistics, such as principal component analysis, will be used to observe relationships among gene expression, metabolite production, root exudation, metal solubility and plant health as affected by NP treatments. Other multivariate analyses such as pairwise comparisons will also be used. One-way analysis of variance will be used to describe treatment effects on selected variables. Geochemical modeling (MINTEQ) of pore water chemistry will provide estimates on the extent of metal complex formation and how that relates to plant uptake and plant response.Outreach efforts include formal training of graduate and undergraduate scholars, as well as high school students participating in summer research programs at USU. A summer research program serving Native American Students at USU Eastern, Blanding Campus, will continue as part of this research.

Progress 06/01/17 to 05/31/18

Outputs
Target Audience:Through dissemination at multiple conferences the target audiences include: Academics, Government, Industry, General Public. Changes/Problems:We are investigating directly treating seeds with the nanoparticles as an alternative approach to dispersing the particles in the growth medium (sand or soil). Preliminary experiments are focusing on influence on seed germination and emergence of endophytes from the treated seeds. What opportunities for training and professional development has the project provided?Three graduate students (2 PhD and one MS) in Biological Engineering have been recruited and trained to work on specific aspects of the research. Three graduate students working on this project (Matthew Potter, MS, Anthony Cartwright PhD, and Shuo Chen, PhD) received interdisciplinary training and professional development from the PI and CoPIs in the areas of nanoparticle synthesis, characterization, surface chemistry and capping layers, and microbiology. Two graduate students (Potter, Cartwright) and one undergraduate (Christina Morgan) attended and presented at a Gordon Research Seminar (GRS) on Nanoscale Science and Engineering for Agriculture and Food Systems. This was transformational for the undergraduate student, who is a first-generation student from the Navajo Nation. One of the PhD students on this project (Anthony Cartwright) received formal training through the USU Microscopy Core Facility to operate the scanning electron microscope, and he is attending a short course / workshop on transmission electron microscopy through the University of Utah. The graduate students working on this project have each supervised 1-2 undergraduate students, training them in fundamental techniques such as nanoparticle synthesis, capping, and characterization, as well as plant growth and microbiology methods, thus providing a highly interdisciplinary training applied toward improving agricultural output under constraints of water stress. How have the results been disseminated to communities of interest?This research has been primarily disseminated through presentations (oral and poster) at professional conferences: Sustainable Nanotechnology Organization, Institute of Biological Engineers, and a Gordon Research Conference co-chaired by the PI. Key findings were also reported in a publication, "Interactions between a Plant Probiotic and Nanoparticles on Plant Responses Related to Drought Tolerance" published in Industrial Biotechnology in June 2018 Our research has also been shared through two summer outreach programs. We hosted two students through the "Native American STEM Mentorship Program" (NASMP) for one week in June 2018. The PI labs also hosted three high school students through the "Biotechnology Summer Academy" organized by Utah State University's Center for Integrate Biosystems for one week in July 2018. This provided opportunities to share the research with these high school students, who investigated microbe-plant interactions and resulting biofilm formation using scanning electron microscopy. These students presented their findings to the other Summer Academy participants, thus disseminating the results to a young audience of future STEM-researchers. What do you plan to do during the next reporting period to accomplish the goals?The capping procedures that have been optimized for the SiO2 NPs will be adapted for the CuO and ZnO particles. Column transport experiments are planned for native and capped NPs in sand and soil columns to assess transport, sorption, and resistance to agglomeration in agriculturally relevant conditions. These particles will then be assessed for activity using wheat with and without PcO6 grown in sand, followed by wheat in the selected Millville soil series. The porewater chemistry from these soils will be fully characterized, and interactions with the NPs assessed, such as formation of natural capping layers on the NPs and subsequent influence on NP activity, availability, agglomeration, and dissolution. A full assessment of NP-induced changes in wheat stomatal conductance and chlorophyll production will be performed, along with assessment of changes in plant morphology and mass. Continued summer outreach education and training will parallel these research activities.

Impacts
What was accomplished under these goals? Accomplishments toward the primary objectives of this research are outlined below: Objective-1. Identify NP-dose relationships that prime plants for drought protection The major activities completed include identifying toxicity threshold levels for CuO, ZnO, SiO2 nanoparticles to the beneficial soil microbe (PcO6) for the uncapped NPs. SiO2 NPs capped with osmolytes of glycine betaine, proline, and arginine were also assessed against PcO6. These background experiments have demonstrated that the capped and uncapped silica NPs are non-biocidal to PcO6 biofilms, in contrast to ZnO and CuO NPs, which exhibit well-defined toxicity thresholds. Using SEM with EDS the SiO2 NPs are observed to be uniformly incorporated within a PcO6 biofilm grown using a root-mimetic hollow fiber membrane to assess NP-biofilm interactions. The specific objectives of identifying the influence of capped and uncapped NPs on wheat response to water stress are ongoing. Reproducible water stress procedures have been developed for wheat grown in sand and will be applied toward achieving this goal. Objective-2. Optimize NP activity / bioavailability through NP coatings The major activity completed was the development of a series of nanoparticle (NP) capping procedures to reduce agglomeration and improve NP availability as assessed through reduction in capped-NP aggregation / precipitation. Specific objectives met include introducing small molecule osmolytes during NP synthesis in order to modify the NP surface charge and chemistry to prevent NP agglomeration. Osmolytes include glycine betaine, proline, and arginine, with the latter selected as a weak base for tuning NP size during synthesis. These osmolyte capping agents have been investigated for commercial and synthesized silicon dioxide NPs. Preliminary findings have demonstrated that incorporation of the capping layers on/in the SiO2 NPs during synthesis provides a more robust capping layer than post-synthesis addition of osmolytes. Agglomeration assays of the native and capped particles have demonstrated a reduced aggregation rate for glycine betaine modified SiO2 NPs. The biological activity of the capped SiO2 NPs has been investigated against biofilms of a beneficial root-colonizing microbe, PcO6 and contrasted with the activity of uncapped NPs of CuO and ZnO. These results have been included in a publication, "Biofilms benefiting plants exposed to ZnO and CuO nanoparticles studied with a root-mimetic hollow fiber membrane." Journal of Agricultural and Food Chemistry. The effect of the NPs on wheat growth and priming against water stress has been investigated for sterile and PcO6-colonized wheat. Wheat grown in sand amended with CuO NPs exhibited greater lignification and resistance to lodging, and these results have been included in "Interactions between a Plant Probiotic and Nanoparticles on Plant Responses Related to Drought Tolerance" published in Industrial Biotechnology in 2018. A portable photosynthesis instrument (LICOR 6800), acquired through a competitive intramural grant, has been used to measure chlorophyll production and gas exchange. Objective-3. Identify the protectant pathways in the plant and rhizosphere primed by the NPs. These efforts are scheduled for years 2-3.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Bonebrake, M., Anderson, K., Valiente, J., Jacobson, A., McLean, J. E., Anderson, A. J., & Britt, D. W. (2017, September 19). Biofilms Benefiting Plants Exposed to ZnO and CuO Nanoparticles Studied with a Root-Mimetic Hollow Fiber Membrane. Journal of Agricultural and Food Chemistry, 66(26), 66196627.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Potter, M. (Presenter & Author), Doxie, S. (Author Only), McLean, J. E. (Author Only), Anderson, A. J. (Author Only), Jacobson, A. (Author Only), Britt, D. W. (Author Only), Sustainable Nanotechnology Organization, "CuO Nanoparticle Modified Lignification of Wheat," Los Angeles, California. (November 5, 2017 - November 7, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Britt, D. W. (Presenter & Author), Mohammad Giasuddin, A. B. (Author Only), Sustainable Nanotechnology Organization, "Self-assembly of tri-functional and di-functional alkane silanes into hydrophobic silica nanoparticles in aqueous media," Los Angeles, California. (November 5, 2017 - November 7, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Britt, D. W. (Presenter & Author), Potter, M. (Author Only), Anderson, A. J. (Author Only), Villanueva, I. (Author Only), Taylor, T. A. (Author Only), Sustainable Nanotechnology Organization, "Summer education in nano- and biological approaches to protect plants against drought stress," Los Angeles, California. (November 5, 2017 - November 7, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Giasuddin, A. B. M. (Presenter & Author), Harris, T. (Author Only), Lewis, R. (Author Only), Britt, D. W. (Author Only), Institute of Biological Engineering, "Aqueous synthesis of silica-spider silk nano-composite materials," IBE, Salt Lake City. (March 30, 2017 - April 1, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Presentations Bonebrake, M. (Presenter & Author), Anderson, K. (Author Only), McLean, J. E., Jacobson, A., Anderson, A. J., Britt, D. W., Institute of Biological Engineering, "Characterization of biofilms and metabolites in a synthetic rhizosphere," IBE, Salt Lake City. (March 30, 2017 - April 1, 2017)