Source: WAKE FOREST UNIVERSITY submitted to NRP
HORMONAL REGULATION OF TOMATO ROOT ARCHITECTURE DURING IRON DEFICIENCY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1003835
Grant No.
2014-67012-22405
Cumulative Award Amt.
$149,688.00
Proposal No.
2014-01972
Multistate No.
(N/A)
Project Start Date
Sep 1, 2014
Project End Date
Aug 31, 2017
Grant Year
2014
Program Code
[A7201]- AFRI Post Doctoral Fellowships
Recipient Organization
WAKE FOREST UNIVERSITY
1834 WAKE FOREST ROAD
WINSTON SALEM,NC 27109-8758
Performing Department
Crop and Soil Sciences
Non Technical Summary
Iron (Fe) deficiency anemia, the most prevalent form of micronutrient deficiency, reduces cognitive performance and immunity. Plants provide the bulk of dietary Fe for the majority of the world's population. Therefore understanding how plants utilize Fe uptake to improve Fe density in plant tissue has important agricultural implications. Arabidopsis has been used as a model for Fe deficiency studies, yet the opposite root morphology responses between Arabidopsis and at least one crop species, tomato, suggests studies of crop plants are needed. This project will test the hypothesis that Fe deficiency-induced changes in flavonoid and ethylene levels contribute to altered lateral root formation and enhanced iron uptake in tomato. This project will utilize tomato mutants with alterations in ethylene signaling and flavonoid synthesis to ask if these signaling molecules are essential for enhanced Fe uptake and morphological changes in roots, using biochemical and molecular biology methods. The role of reactive oxygen species in response to Fe deficiency and flavonoid action as antioxidants will be explored.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2031460101050%
2031460105050%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1010 - Nutrition and metabolism; 1050 - Developmental biology;
Goals / Objectives
Scientific goals/objectives: the overall goal of this project is to determine to what extent the roles of hormones (ethylene and auxin) and flavonoids play in modulating root architecture in response to iron (Fe) deficiency in tomatoes. Objective 1: Does Fe deficiency influence ethylene synthesis in tomato roots, and is ethylene required for the transcription of necessary Fe uptake genes? Objective 2: Does increased ethylene synthesis in Fe-deficient tomato roots induce flavonoid synthesis? Objective 3: Does Fe deficiency generate reactive oxygen species (ROS) in tomato roots, and are flavonoids synthesized during Fe deficiency used as an antixoidant to combate toxic effects of ROS? Objective 4: Fe deficiency has been shown to result in opposing effects of lateral root (LR) growth between Arabidopsisand tomato:ArabidopsisLR formation is reduced, while tomato LR formation increases. The hormone auxin promotes LR growth in both Arabidopsisand tomato, while ethylene and flavonoids suppress auxin. The opposing LR phenotypes inArabidopsisand tomato in response to Fe deficiency suggests that auxin, ethylene, and flavonoids are regulated differently in these plants' response. Objective 4 will look at the transport rate/ability, responsiveness and transcription of auxin-related genes in tomato. These four objectives will then be combined in order to determine how ethylene and flavonoids modulate auxin in tomato roots during Fe deficiency to increase LR formation.Professional goals/objectives: a large part of this project concentrates on my professional goal of becoming a professor. The scientific objectives will allow me to execute and manage an independent project while pursuing a new avenue of research in my current laboratory. My advisor, Dr. Gloria Muday, will oversee my progress in creating a name for myself in the scientific community, as well as helping me hone my teaching skills. I will participate in national meetings, and collaborations to connect me to other scientists. I will also join classes offered at Wake Forest University and help teach classes taught by Dr. Muday.
Project Methods
METHODS:1) Determine transcription levels of necessary iron (Fe) uptake genes in tomato mutants with defects in hormonal/metabolite production/signaling using quantitative reverse-transcriptase PCR (qRT-PCR) under Fe deficient conditions. Transcriptional levels will be determined from plants grown hydroponically.2) Determine if Fe deficiency alters lateral root (LR) phenotypes in tomato mutants grown in agar. This method will employ opaque plates where tomato shoots are exposed to air.3) Flavonoid biosynthesis of tomato mutants will be assesed during Fe deficiency: transcriptional levels of key flavonoid biosynthesis genes will be determined by qRT-PCR, flavonoid levels will be measured by liquid chromatography-mass spectrometry (LC-MS), and ethylene levels will be measured using traditional methods.4) Fe levels in roots and shoots will be measured using inductively coupled plasma mass spectrometry (ICP-MS).5) Reactive oxygen species (ROS) will be measured using fluorescent dyes, and transcriptional levels and activity of ROS scavenging proteins will be measured by qRT-PCR and activity by traditional methods. 6) Auxin responsiveness will be measured by using a well-known transgenic plant harboring a DR5-GUS construct. Auxin transport will be determined using radioactively-tagged synthetic auxin and free IAA levels measured.EFFORTS:Laboratory instructionEVALUATION:Data collected: quantititative measurement of gene transcription levels, Fe levels, auxin levels, and LR phenotypes.Milestones: completion of objectives and publication of data, presentation of data at national scientific conferences and PD meetings in Washington, DC.

Progress 09/01/14 to 08/31/16

Outputs
Target Audience:Target audiences reached by this project include scientists, graduate students, and post-doctoral researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided a wealth of professional development. PD attended several workshops at the 2016 Plant Biology conference run by the American Society of Plant Biologists. There, PD was able to network wiht young scientists and assistant professors to gain insight in improving ongoing experiments related to this project and in planning for future employment. Additionally, the teaching opportunities outlined in the last reporting period has prepared PD to teach a new course to talented 7th and 8th graders,where plant biology and environmental sciences was a topic. This year has also encompassed further training for PD on LCMS and ICP-MS techniques. How have the results been disseminated to communities of interest?Results have been shared at a national conference, Plant Biology 2016. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the course of this project, we were able to resolve Objective 3 (Does Fe deficiency genearate ROS in tomato roots) and assess its relation to flavonol content in roots.

Publications


    Progress 09/01/14 to 08/31/15

    Outputs
    Target Audience:Members of the scientific community were informed of my project goals and outputs at several conferences attended during this period.Additionally, high school students that participated in a genetics workshop were also reached. These students were guided through basic Mendialian genetics in order to learn how traits were inherited in plants. These concepts were then applied to learning desirable traits in plants that enhance drought tolerance and techniques common to traditional plant breeding and genetic modification. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities during this reporting period include guest lectures an introductory biology class and Plant Physiology at Wake Forest University. I have participated in outreach activities that center on teaching genetics to local high schools. Professional development activities include attendance at several conferences (CMCS at Wake Forest University, PMB for plant biologists in NC, and the PD Fellow's meeting at Washington DC).I have also participated in several professional development courses at Wake Forest University, as well as those offered at Elon University. How have the results been disseminated to communities of interest?Current results have been presented at several conferences (CMCS at Wake Forest University, PMB for plant biologists in NC, and the PD Fellow's meeting at Washington DC). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Iron (Fe) deficiency anemia is the most prevalent form of micronutrient deficiency worldwide. Dietary Fe for the majority of the world's population is obtained from plants.Results obtained from this project will help researchers understand the intricate hormonal networks involved in regulation of plant development in response to Fedeficiency. This project translates findings in the model plant,Arabidopsis, to the economically important crop, tomato. As Arabidopsis and tomato have been reported to have opposing changes in root structure in response to Fe deficiency, further research in the Fe deficiency response ofcrop plants are needed. Accomplishments: Objective 1: Does Fe deficiency influence ethylene synthesis in tomato roots, and is ethylene required for the transcription of necessary Fe uptake genes? 1) Major activities completed / experiments conducted: 1 - standardization of growth conditions. 2) Data collected: RNA for qPCR analysis collected. 3) Summary statistics and discussion of results: none reported 4) Key outcomes or other accomplishments realized: standardized hydroponic growth conditions for future experiments; taught undergraduate and graduate students how to grow tomatoes hydroponically. Objective 2: Does increased ethylene synthesis in Fe-deficient tomato roots induce flavonoid synthesis? 1) Major activities completed / experiments conducted: flavonol and anthocyanin quantification of hypocotyls and leaves. 2) Data collected: flavonol and anthocyanin quantification of hypocotyls and leaves. 3) Summary statistics and discussion of results: Fe deficiency increases flavonoid synthesis in hypocotyls and leaves. 4) Key outcomes or other accomplishments realized: none reported Objective 3: Does Fe deficiency generate ROS in tomato roots and are flavonoids synthesized during Fe deficiency used as an antioxidant to combat toxic effects of ROS? 1) Major activities completed / experiments conducted: visualization of total ROS or superoxide accumulation in tomato roots. 2) Data collected: images of ROS accumulation in tomato roots. 3) Summary statistics and discussion of results: Fe deficiency increases ROS in tomato roots. 4) Key outcomes or other accomplishments realized: none reported Objective 4; Does Fe deficiency affect the transport rate/ability, responsiveness, and transcription of auxin-related genes? 1) Major activities completed / experiments conducted: none reported 2) Data collected: none reported 3) Summary statistics and discussion of results: none reported 4) Key outcomes or other accomplishments realized: none reported

    Publications

    • Type: Book Chapters Status: Awaiting Publication Year Published: 2015 Citation: Gayomba, S.R., Watkins, J.M., and Muday, G.K. Flavonols regulate plant growth and development through regulation of auxin transport and cellular redox processes. Recent Advances in Polyphenols Research.