CHARACTERIZATION OF THE ROLE OF THE MEDICAGO TRUNCATULA GIRAFFE GENE IN OXIDATIVE STRESS.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0227489
• Project No.: VT-H01809
• Project Start Date: Oct 1, 2011
• Project End Date: Sep 30, 2014

Project Director
Harris, J.

Recipient Organization
UNIVERSITY OF VERMONT
(N/A)
BURLINGTON,VT 05405

Performing Department
Plant Biology

Non Technical Summary
Plants are rooted to one spot for their entire life. In order to survive, plants must sense even the smallest environmental change, and respond rapidly, changing their physiology and modifying further development. Understanding the rapid molecular changes that underlie plants responses to their environment can help us to optimize conditions or treatments that maximize plant yield under stressful conditions. Oxidative stress is a physiological process that is common to both plants and animals. It involves the production of reactive oxygen species (ROS) in response to environmental stresses. In plants, oxidative stress is a response to both abiotic and biotic stress; in other words, it is a response to the stresses caused both by non-living and living parts of the plants local environment. Cold, drought, high light, UV, high salt, wounding, pathogen attack and heavy metals are all environmental stresses that induce oxidative stress in plants. Oxidative stress was originally thought of as a stress that should be avoided. Now ROS are thought of as key signaling molecules that can be generated rapidly, in response to an environmental stress or developmental event, and then are just as quickly destroyed. In this way ROS molecules can function as a rapid response to an environmental stress, triggering larger scale changes in the cell that ultimately result in systemic responses throughout the plant. We are interested in understanding how plants respond to environmental stress and how the production and response to ROS is regulated. Our project focuses specifically on the role of the heme oxygenase enzyme and the hormone abscisic acid in the production of ROS and the response to ROS signals during root growth and the formation of nitrogen-fixing root nodules.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	2420	1020	15%
203	2420	1080	70%
206	1413	1020	15%

Knowledge Area
206 - Basic Plant Biology; 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1413 - Peas (fresh, fresh-processed); 2420 - Noncrop plant research;

Field Of Science
1080 - Genetics; 1020 - Physiology;

Keywords

reactive oxygen species

oxidative stress

abscisic acid

heme oxygenase

medicago truncatula

root

nodule

latd

Goals / Objectives
The objective of this proposal is to test the hypothesis that the GIRAFFE gene is involved in ROS homeostasis in Medicago truncatula and to ask whether it functions in roots and nodules in concert with the LATD gene, another ROS-regulating gene. OBJECTIVE 1: Is the Heme Oxygenase putatively encoded by the GIRAFFE gene involved in ROS homeostasis in Medicago truncatula roots and nodules The function of Heme Oxygenases in plants is still very much a matter of discussion. In our first objective, we plan to test the hypothesis that the GIRAFFE gene is involved in regulating the levels of ROS. In particular, we will test whether plants lacking a functional GIRAFFE gene have abnormal levels of ROS and whether ROS levels regulate expression of the GIRAFFE gene. OBJECTIVE 2: Do LATD and GIRAFFE function in a common pathway to regulate ROS in Medicago roots The LATD gene characterized in my lab regulates ROS levels in roots. Here we are testing whether GIRAFFE functions in the same pathway as LATD. We will examine whether mutations in either LATD or GIRAFFE affect expression of the other gene. OBJECTIVE 3: LATD functions in an abscisic acid (ABA) signaling pathway in roots. Does GIRAFFE function in an ABA signaling pathway also In these experiments we will test whether GIRAFFE and ABA function in a common regulatory pathway. The following experiments test whether giraffe mutants are still sensitive to ABA and whether ABA turns the GIRAFFE gene ON or OFF.

Project Methods
Plant Growth conditions: For all experiments, M. truncatula plants will be grown on BNM agar, pH 6.5. Seeds are scarified, surface sterilized and germinated in moist, sealed petri dishes in the dark as described, prior to plating on the sterile agar. For most experiments, the germinated seedlings will be grown on BNM agar in large plates (245 x 245 mm), but experiments done with young seedlings will be done on smaller square plates. Where needed, Abscisic Acid will be added to the medium after autoclaving to achieve a final concentration of 1 or 10 micromolar. Plates will be sealed with surgical tape and placed in a vertical position, so that the roots grow down the surface of the agar. For nodulation assays, plants will be flood inoculated at Day 3 with a washed suspension of Sinorhizobium meliloti strain Rm1021 in 10 mM magnesium sulfate. Nodules are scored by eye at 14 or 17 days post-inoculation. For adding ROS, plants will be grown in growth pouches with liquid BNM and H2O2 or ROS inhibitors will be added directly to the medium. Growth chamber conditions for all plants are 20 degrees C, 40% humidity, with approximately 120 micromoles/m2 sec of white light given with a 16 hour day/8 hour night cycle. Gene expression analysis: RNA will be extracted using the Trizol method, and treated with DNAse to remove contaminating genomic DNA. RNA quality will be checked at the UVM DNA facility and RNA that passes the quality control will be converted into cDNA using a kit from Invitrogen. This cDNA will then be used as the basis for qRT-PCR analysis using a new ABI machine. We will use the SYBR green protocol and will analyze using the deltaCT method. All analyses will be normalized to two housekeeping genes and analyzed for statistical significance using ANOVA.

Progress 10/01/11 to 09/30/14

Outputs
Target Audience:Oxidative stress is important step in the plant response to most abiotic and biotic stresses. Plant physiologists are interested in understanding how this process works. Identifying genes that modulate the production or scavenging of reactive oxygen species could provide potential targets for plant breeders. Currently, the audience is other plant biologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two students, one graduate and one undergraduate, have learned from participating in this project. The graduate student, Parna Ghosh, presented her work as a poster at two national meetings. She is now employed by the Noble Research Foundation based on her Masters research project funded by Hatch. How have the results been disseminated to communities of interest?The results of this project have been presented at national meetings (ASPB, 2012 and the North American Symbiotic Nitrogen Fixation Conference, (July, 2013). A manuscript is in preparation. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We found that GIRAFFE regulates the expression of genes encoding enzymes involved in ROS production or scavenging. Genes regulated encode the NADPH oxidase, RBOHC, and a putative cell wall peroxidase, cwPRX2, in both roots and shoots. Since both of these genes are also regulated by the LATD/NIP gene, we examined whether GIRAFFE and LATD/NIP regulate each other's expression. We showed that the expression of neither LATD/NIP nor GIRAFFE are dependent on the other, even though they regulate the expression of a common set of genes, indicating that they likely function in different pathways. We also found that the GIRAFFE gene functions during nodule senescence, and is required then to maintain high levels of both RBOHC and RBOHE.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, Chang, Bousquet, Amanda and Jeanne Marie Harris. (2014) Abscisic acid and LATD/NIP modulate root elongation via reactive oxygen species in Medicago truncatula. Plant Physiology, 166(2):644-658.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: My target audience at this stage is the scientific community interested in nodulation and in oxidative stress. In addition, one undergraduate and one graduate student were trained as a part of this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Parna Ghosh is a masters student who worked on this project. Parna is examining regulation of the expression of enzymes required for reactive oxygen species homeostasis. She learned experimental design, the skills needed for studying RNA expression by quantitative RT-PCR, and analysis of nodulation. Beck Powers is an undergraduate student who received a summer research grant from UVM to participate on this project. She has also continued her project during the school year. Beck has been investigating the role of the heme oxygenase enzyme in cadmium resistance. She presented her work at a local scientific workshop. How have the results been disseminated to communities of interest? We had an abstract and poster at a national meeting this past summer (North American Symbiotic Nitrogen Fixation Conference, Minneapolis, MN, July 2013). In addition, Parna Ghosh (Masters student) gave a brief oral presentation on the results from this project at the New England Sinorhizobium-Medicago workshop in October, 2013. What do you plan to do during the next reporting period to accomplish the goals? We are currently testing whether the GIRAFFE heme oxygenase regulates expression of genes involved in oxidative stress in nitrogen-fixing nodules when they senesce.

Impacts
What was accomplished under these goals? We found that the GIRAFFE heme oxygenase regulates the expression of three NADPH oxidase genes in the root and the shoot. We found that it regulates a subset of the LATD-regulated ROS-related genes. However, we found that GIRAFFE does not regulate LATD gene expression, nor is its expression regulated by LATD, suggesting that they may function in parallel pathways.

Publications

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: The purpose of this project is to determine the role of the Medicago truncatula heme oxgenase enzyme in root growth and the development and functioning of symbiotic root nodules. We have found that this heme oxygenase enzyme regulates the expression of certain enzymes involved in the generation or scavenging of reactive oxygen species. We are currently testing whether the heme oxygenase is involved in resistance to the heavy metal, cadmium, and whether it is involved in the aging of nitrogen-fixing root nodules. We have presented this work in scientific meetings. PARTICIPANTS: Parna Ghosh is a masters student who worked on this project. Parna is examining regulation of enzymes required for reactive oxygen species homeostasis. She learned the skills needed for studying RNA expression and presented her work at a national meeting. Beck Powers is an undergraduate student who received a summer research grant from UVM to participate on this project. She has also continued her project during the school year. Beck has been investigating the role of the heme oxygenase enzyme in cadmium resistance. She presented her work at a local scientific workshop. TARGET AUDIENCES: Two students, one graduate and one undergraduate, have learned from participating in this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have found that the GIRAFFE heme oxygenase gene alters the expression of certain enzymes that are required for oxidative stress signaling. This research helps to extend our understanding of how reactive oxygen molecules signal during normal development.

Publications

• No publications reported this period