UNDERSTANDING THE PHYSIOLOGY AND GENETICS OF CARBON TRANSPORT TO MAIZE CROWN ROOTS DURING DROUGHT STRESS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1026444
• Grant No.: 2021-67034-35142
• Cumulative Award Amt.: $120,000.00
• Proposal No.: 2020-09933
• Project Start Date: Jun 15, 2021
• Project End Date: Jun 14, 2024
• Grant Year: 2021
• Program Code: [A7101]- AFRI Predoctoral Fellowships

Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211

Performing Department
Plant Sciences

Non Technical Summary
This research project focuses on understanding how carbon transport to roots affects root growth andoverall drought tolerance.Previous research has shown that the maize nodal root system, aroot type unique to grasses (including cereal crops) that supplies the bulk of water to theplant, are unique among plant tissues in their capacity for continued growth in dry soil. Thecentral hypothesis in this project is that continued nodal root growth is maintained byincreased carbon transport to the nodal roots during water deficit. This project aims todevelop a better understanding of the physiology of carbon transport during drought stress,and identify genes involved in root growth and carbon transport during drought. Progress inthese areas will contribute greatly to plant health and production as well as contribute to thePD's educational and professional development.The two main objectives of the proposed research are: 1) Characterize carbon transport tonodal roots during precisely-controlled drought conditions; 2) identify genes contributing toroot growth and carbon transport to roots during drought. For the first objective, I will utilizea specialized growth apparatus that imposes precise drought treatments, and I will develop anexperimental system to measure carbon transport within the plant. For the second, I willleverage existing datasets to identify potential genes regulating carbon transport and rootgrowth, and the carbon transport-defective mutant Zmsut1, to explore genetic control ofcarbon transport to roots and drought tolerance. This project uses Maize but findings arelikely to be translatable to other crops.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	1510	1060	100%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1510 - Corn;

Field Of Science
1060 - Biology (whole systems);

Keywords

carbon transport

drought stress

maize

Goals / Objectives
With global population expected to reach 10 billion by 2050, agricultural productivity is increasingly important. Water is the number one limitation on agricultural yield world-wide. To meet the demands for food, fiber, and plant-based fuels, we must improve the ability of plants to reach and take up water. The uptake and acquisition of water is mediated by plant roots; therefore, we must understand the traits governing and contributing to root growth, particularly in the arid conditions that will likely characterize agriculture in the coming decades. The root system of grasses includes an embryonic root system, which supplies water during seedling establishment, and a system of stem-borne nodal roots, also referred to as "crown roots". In maize, nodal roots supply most of the water after seedling establishmentand deeper nodal root growth is a key adaptation to retrieve water in dry soil. Nodal roots are unique among plant organs for their capacity to maintain growth at water potentials (ψw) low enough to completely cease growth of other plant tissues. Deciphering the mechanisms that contribute to this incredible capacity for tissue expansion in dry environments will be crucial for plant breeding efforts aimed at improving drought tolerance.When growing in a low water potential environment, such as dry topsoil, continued root growth requires an increased concentration of carbon-based solutes, such as proline and sugars. Increased solute concentration helps expanding cells maintain turgor and expansive growth.To facilitate this, plants re-prioritize carbon resource allocation when experiencing water deficit (WD). Understanding the relationship between carbon supply, WD stressed roots, and how roots maintain growth is critically important for understanding plant responses to drought. Elucidating the physiological mechanisms that control this process will lead to major advances not just in maize, but potentially all cereal crops. Identifying the genes underlying these mechanisms will provide targets for future crop breeding efforts.This proposal will further develop my skills as a researcher and prepare me for a career leading research projects in industry, academic, or government research institutions. The major scientific goal is to develop a mechanistic understanding of how carbon partitioning in maize nodal roots relates to their ability to maintain growth during drought stress. Specifically, I will determine if increased carbon flux to the nodal root tip is required to maintain growth during WD, and if carbon partitioning to sink tissues changes in response to WD stress. I will also identify putative regulators of nodal root growth maintenance during drought stress using previously identified genes from an RNA-seq study. To test my hypotheses, I will address the following objectives:1) Characterize carbon flux to maize nodal roots during precisely-controlled drought regimes using a split-chamber growth system and isotopic 14CO2 radiotracer, and 2)Test the roles of genes identified by a drought stress RNA-seq experiment in nodal root growth and drought tolerance using genetic mutants.

Project Methods
Efforts1. Characterizing carbon flux to maize nodal roots during precisely-controlled droughtregimes and using a split-chamber growth system and isotopic 14CO2.Imposing precise and repeatable drought treatments on maize nodal rootsThis system has been designed and validated for analyzing nodal roots emerging from thesecond whorl of the crown and represents a highly specialized approach to studying nodalroot growth and physiology. Preliminary kinematics studies found noshortening in the growth zone of nodal roots in the second whorl for water potentials as low as -2MPa (manuscript in preparation). As it was determined that nodal root elongation in the second whorl (cv. FR697) is completely maintained in this system, I will use this cultivar and the second node of crown roots as a model system in the split chamber apparatus. Four conditions will be used, three of which represent steady-state water relations, as evidenced by uninhibited shoot growth, which is more sensitive to low water potentials than root growth. For these three treatments, primary and seminal roots will be grown in a WW inner chamber, whereas nodal roots will be grown in an outer chamber with water potentials of -1.8MPa, -0.9MPa, and ≥-0.1MPa (field capacity). This design will manipulate the sink strength (i.e. carbon demand) of the growing nodal roots by manipulating the water potential of soil they will be exposed to, as lower ψw requires more solute deposition to maintain turgor. The fourth treatment will use the previously described condition, wherein the ψw of the inner chamber (supplying the primary and seminal roots) will be -0.4MPa, which we demonstrated to substantially reduce shoot growth and result in a classical drought stress phenotype. I have performed many experiments using this phenotyping system, including generating drought stressed root tissue used in the RNA-seq experiment.Quantifying carbon partitioning to nodal roots during drought stressTo quantitatively measure carbon fluxes to nodal roots and other sink tissues, I will use a proven pulse-chase method. In a sealed chamber, I will administer 14CO2 to plant replicatesexposed to each stress treatment using the split chamber apparatus. Then, I will measure 14C accumulation in harvested tissues using published methods.2. Test the roles of genes identified by drought-stress RNA-seq experiments in carbontransport to nodal root growth and drought tolerance using genetic mutants.Build reverse-genetics tools for investigating candidate genes that regulate root growthIn a previous experiment, I used the split-chamber growth apparatus and an RNA-seq approachto survey changes in gene expression associated with drought stress in the growth zone of maize nodal roots. To gain insight into the biological functions affected by this large suite ofdifferentially expressed genes, I have begun preliminary bioinformatics analysis using geneontology (GO) and KEGG pathway enrichment. Primary metabolism, secondary metabolism, and carbon metabolism are among the most commonly represented pathways, suggesting specific changes to carbon transport and utilization in the growing region of nodal roots are required to maintain growth during WD. To test this hypothesis, I am acquiring insertional mutant alleles for candidate genes identified by this RNA-seq experiment and am backcrossing them into the genetic background (cv. FR697) that was used when developing our split-chamber growth system. To date, I have leveraged public germplasm resources [22-24] to obtain mutant alleles for 43 unique genes that had significantly altered gene expression (cut-off of log2 fold change ≥ 2) in the growth zone of the nodal root tip during drought stress. I narrowed this list of genes by selecting only those with the highest nominal expression in nodal roots, based on public data. The resulting genes are diverse in class and putative function. Further analysis of this dataset under guidance of collaborating mentor Joshi will facilitate better support of likely gene candidates and eliminate unlikely candidates by better leveraging public data to build more robust hypotheses and save research time. This experience will enhance my skillset by providing essential training in computational biology approaches. Building a collection of mutant lines based on genes identified in this data set will let me leverage my existing data to test hypotheses about the roles of these genes in carbon transport to roots, regulation of root growth, and drought tolerance. Importantly, this effort is built upon Mendelian genetics concepts and uses basic molecular biology techniques, making it a great opportunity to train undergraduate students, providing me further mentoring experience.Screening mutant lines for drought tolerance and root growth phenotypesAfter generating seed stocks that segregate mutant alleles, I will first screen mutant lines in asystem allows rapid screening for root growth and drought-tolerance phenotypes. I will perform these experiments under guidance of Co-Mentor Sharp, who developed this system and possess expertise in crop physiology. I will compare growth and carbon transport to roots in mutants of interest to their wild-type counterparts using the split-chamber growth apparatus and perform 14CO2 pulse-chase experiments to investigate if carbon partitioning to nodal roots underlies the identified drought-tolerance and root growth phenotypes.EvaluationThe PD will work with the primary mentor to evaluate professional goals using the individual development plant (IDP) created together. Project progress will be continuously documented via the IDP. Dr. Braun and I will meet weekly to discuss project strategy, trouble-shooting, and discussion. This process will facilitate continued project success, and also develop my scientific understanding and ability as an independent researcher. Weekly meetings will ensure deliverables outlined above are achieved as stated. Attendance at scientific conferences and social media accounts will disseminate results to the general public. To document my career progress during and beyond the term of this fellowship, I will maintain profiles on established professional social networking sites, such as Linkedin and Plantae. These measures will build on my teaching experience and mentorship of undergraduate students, helping me to develop science communication, mentorship, and project management skills that will enhance my future career as a research leader.

Progress 06/15/21 to 06/14/24

Outputs
Target Audience:The results of this project will be significant to academic researchers and the agricultural industry. Breakthrough knowledge pertaining to genetics governing root growth during drought stress serves the agricultural community, including farmers, when translated to improved crop varieties. Changes/Problems:The most significant departure from the project plan was the decision to not pursue radioisotope based experiments. This was due to the unforseen amount of time requried to get institutional permission to use the protocol, which I had thought we were close to obtaining when the project was proposed. Perhaps more significantly, I was forced to take a leave of absence for an entire year during the project. The leave of absence was coordinated with NIFA administrators who granted a one year extension, but my university did not allow me to continue as a graduate researcher for more than additional semester upon my return. Objective two planned to use genetic mutants to investigate the role of candidate genes identified through RNAseq. While several mutant lines were developed by introgressing publicly available insertional alleles, none of the developed lines were pertinent to major findings. With additional time and funding, transgenics would have been possible, but this exceeds the scope of resources afforded by this project. What opportunities for training and professional development has the project provided?Over the course of this project, I gained new skills and experience in new experimental methods related to plant physiology, biochemistry and biochemical analysis, and data science. This project also afforded me an opportunity to hone soft skills, as I worked with many collaborators to solve challenges. I presented my research at multiple conferences and symposia, which was a valuable experience as well. How have the results been disseminated to communities of interest?I gave a talk on my findings at twomajor conferences, and have drafted and submitted a manuscript to a peer reviewed journal which will detail the findings for other researchers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) Carbon flux to maize nodal roots during drought was evaluated using the split chamber growth system. We designed,built,and tested an isotopic gas phase pulse-chase 14C system but did not utilize isotopic labeling due to technical limitations (to include radioisotope permitting and use challenges) and the significant amount of time required to perform these experiments while the project director's time was limited due to unforseen circumstances (moblization for military service). Instead, carbon flux was investigated by indirect measurements. Frowth parameters and physiometric characteristics were evaluated in multiple drought regimes. Results showed that maize nodal roots forcv. FR697 maintain growth relative to well-watered control in soil as dry as-0.9 MPa. Further experiments determined that neither dry weight nor water content werereduced within the growth zone (apical 1 cm) of the nodal roots. Kinematic analysis of the growth zone showed that it was not shortened during water deficit, relative to well-watered control. In contrast to root growth, shoot biomass was significantly reduced by water deficit. Taken together, the data demonstratethat maize (cv. FR697) maintainnodal root growth in dry soil by repartitioning biomass from developing leaves. 2) Canadidate drought-response genes were identified by RNAseeq using well-watered control and drought treated tissue harvested from the growth zone (apical 1 cm) of nodal roots. The results identified the nature of water deficit response in elongating nodal root tissue and suggested that ABA perception and signal transduction was a response to water deficit. Our RNAseq data identified a novel ABA receptor, PYL7, which appears to be uniquely expressed in nodal roots. During water deficit, ABA content in nodal root tips is elevated fourfold but transcript abundance of PYL7 is significantly attenuated in the same tissue, suggesting that water stressed maize roots coordinate expression of ABA receptors in order to maintain the proper signal transduction and growth responses. We also measured the accumulation of solutes in well-watered control and drought treated nodal root growth zone. Our data showed significant accumulation of osmolytes including betaine and proline, as well as ROS scavenging glutathione. We profiled expression of genes involved in biosynthesis and degradation of these molecules using RNAseq. Integrating our metabolite and transcript datasuggested that during water deficit, maize nodal root tips synthesize proline using the saccharopine pathway rather than the more typical biochemical pathway, and repress catabolism of proline. We attempted to quantify the levels of solutes, including amino acids, in order to compare our data to other studies. This experiment was unscuccesful due to technical reasons.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: "Maize nodal root growth maintenance during water deficit: metabolic acclimation and the role of increased solute deposition in osmotic adjustment" (2024). McCubbin TJ, Greeley LA, Mertz RA, Sen S, Griffith LA, King-Miller SK, Casey K, Niehues ND, Pareek A, Bryan VJ, Zeng S, Ghani A, Joshi T, Peck SC, Oliver MJ, Fritschi FB, Braun DM, Sharp RE. Frontiers in Plant Science.

Progress 06/15/22 to 06/14/23

Outputs
Target Audience: Nothing Reported Changes/Problems:PI is on a leave of absence from the University for military service. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? PI is on a leave of absence from the University for military service.

Publications

Progress 06/15/21 to 06/14/22

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?As stated above,I have drafted and will shortly submit a manuscript describing much of the preliminary data that generated the hypotheses I described above. These experiments aimed to understand how maize responds to drought stress (a more broad question than the first objective described above) and what genes respond to drought stress in nodal roots (the data set described above in objective two). With the support of this fellowship I have been afforded the time and research support to analyze the data from these experiments and draft a manuscript. During the course of this activity I have gained experience and training in bioinformatics techniques, as well as professional experience in academic and scientific writing and project management. During the course of this reporting period I have supervised and mentored and undergraduate student intern who is conducting an independent research project. How have the results been disseminated to communities of interest?At this time, I am drafting a manuscript which will communicate some findings relating to both major objectives. This will be submitted to major peer reviewed journal. When possible, results will be shared at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?The experiments described in the first objective will be performed. This includes conducting the experiments using the described apparatus and radioisotopes as well as analyzing the data. Controlled drought experiments will be performed to determine the role of genes identified from objective two. The data will be analyzed and a manuscript will be drafted.

Impacts
What was accomplished under these goals? Broadly, this project aims to improve our understanding of plantgenetics and physiology in order to improve crop yields in the face of increased drought pressure. This is a major challenge facing the world, as populations continue to grow but the amount of land and water available for agriculture continues to shrink. Moreover, climate change has increased the frequency and severity of droughts across the US and around the world. Meeting these challenges will require shifting priorities in plant breeding, to focus not just on yields under ideal conditions, but in developing crops that can withstand less water availability and support plant growth in drier environments. An ideal way to pursue this goal is to improve the root systems of crops, in order to improve the uptake of water from the soil. The majority of calories in the human diet come from cereal crops, which includes maize (corn), rice, wheat and barley. Cereals are plants that belong to the grass family. Grasses are unique in that their root systems are unlike that of most other plants, including dicotyledenous crops such as soybeans or trees; the typicalnon-grass plant contains a deep taproot with lateral roots eminating from it. The root systems of grasses is referred to as "fibrous" and contains a primary root and seminal roots that growfrom the germinating kernal (seed), which is analogous to a taproot. However, grasses also contain an additional system of roots that grow from the stem, referred to as crown roots or nodal roots. In maize, the oldest of these is referred to as "brace roots" and can be seen growing from the stalk into the ground in mature plants, as one might see in a corn maize. Nodal roots are particularly important because they physically anchor the plant to the ground, preventing lodging, and they provide the majority of water uptake in mature plants. Despite their importance, relatively little is known about the genes governing their growth or how their growth is maintained in drought stress plants. What is known, however, is that nodal roots exhibit a unique ability to continue growing when drought stresed to levels that inhibit the growth of other organs and tissues. Maintaing nodal root growth during drought allows the plant to reach deeper into the soil where water is more available, thereby allowing the plant to withstand the stress of hot, dry environments.The goal of this project is to understand how this continued nodal root growth during drought stress is accomplished at a genetic and biochemical level. By gaining this knowledge, plant breeders will be empowered to pursue traits that will lead to higher performing and more drought resiliant crops, benefitiing farmers, consumers, and the environment. During this reporting period (previous year) work was undertaken in pursuit of both major objectives. The first major obejctive of this research project aims to understand howdrought stressed maize plants allocatecarbohydrates for growth. Specifically, we will test the hypothesis that in order to sustain nodal root growth during drought stress, the plant exports more carbohydrates (the raw material for growth and development, synthesized from photosynthesis) to the nodal roots than it would when not experiencing drought stress. Characterizing the transport of carbohydrates requires using radioactive isotopes to track and measure the export of carbon from the site of photosynthesis in the elaves, to the growing tips of roots. To achieve this, I will use a custom constructed chamber that is air-tight, in which I can place plants and administer radioactive carbon isotopes. I began constucting this apparatus immediately prior to submitting this project proposal, and in the past year have worked with environmental health and safety personnel at my institution to design a safe experimental procedure for these experiments, and tovalidate the safety of the apparatus. This was a necessary prerequisite for conducting these experiments involving radioisotopes and took many months of proposing the methodology and seeking approval from the institutional radiation safety committee, and testing the apparatus. With this hurdle cleared I can begin conducting experiments. The second objective involves leveraging data I gathered from an experiment that identified what genes were expressed in nodal root tips in response to drought stress. This data set could shed light on the genes requried to maintain growth of nodal roots in dry soil, but determing whichgenes are likely contributing to growth out of the many thousands of genes identifying is a difficult task. During this reporting period, I analyzed this data set with the help of my collaborating mentor, Dr. Joshi, who is an expert in bioinformatics. This led to identifying several genes that might be contributing to nodal root growth and drought stress tolerance. To test these hypotheses, I obtained germplasm with putative mutant alleles for the genes of interest. This will enable experiments to determine what role these genes play in root growth during drought, by growing normal ("wild-type") plants along side plants that are otherwise genetically identical, but contain mutant (non-functional) versions of these genes of interest. If the mutants do not maintain nodal root growth comensurate with the wild type plants either consitutively or during drought stress, we can conclude that these genes contribute to nodal root growth. These types of experiments have the potential to generate very useful data for plant breeders. During this reporting period, I have conducted preliminary analysis of mutant germplasm in the greenhouse and in the field. I have identified one gene is key for normal plant growth and development, and five genes which may contribute to drought stress tolerance. Controlled experiments will be necessary to assess the roles of these genes in root growth during drought stress. In addition, I have drafted and will shortly submit a manuscript describing much of the preliminary data that generated the hypotheses I described above. These experiments aimed to understand how maize responds to drought stress (a more broad question than the first objective described above) and what genes respond to drought stress in nodal roots (the data set described above in objective two). With the support of this fellowship I have been afforded the time and research support to analyze the data from these experiments and draft a manuscript. This will contribute a change in knowledge in the field.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Wang, J., Sidharth, S., Zeng, S., Jiang, Y., Chan, Y. O., Lyu, Z., ... & Joshi, T. (2022). Bioinformatics for plant and agricultural discoveries in the age of multiomics: A review and case study of maize nodal root growth under water deficit. Physiologia Plantarum, 174(2), e13672.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Julius, B. T., McCubbin, T. J., Mertz, R. A., Baert, N., Knoblauch, J., Grant, D. G., ... & Braun, D. M. (2021). Maize Brittle Stalk2-Like3, encoding a COBRA protein, functions in cell wall formation and carbohydrate partitioning. The Plant Cell, 33(10), 3348-3366.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: McCubbin, T. J., & Braun, D. M. (2021). Phloem anatomy and function as shaped by the cell wall. Journal of Plant Physiology, 266, 153526.