A NODULE REGULATORY NETWORK INVOLVING LIGHT, ETHYLENE AND JASMONIC ACID

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0215219
• Project Start Date: Oct 1, 2008
• Project End Date: Sep 30, 2011
• Program Code: [(N/A)]- (N/A)

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

Performing Department
PLANT BIOLOGY

Non Technical Summary
Legumes form a symbiosis with soil bacterium, collectively known as Rhizobium, that results in the formation of nitrogen-fixing nodules on the root of the host plant. The fixed nitrogen produced in these nodules acts as an internal source of fertilizer for the plant, and enables legumes to grow vigorously even in nitrogen-poor soils. Environmental conditions have profound effects on the ability of a plant to nodulate. In order to develop sound agricultural practices, we must increase our understanding both of the effects of different environmental conditions as well as the mechanism of the plant response. Light is one of the most important aspects of a plant?s environment. Preliminary data from our lab indicates that different wavelengths of light have the ability to increase or decrease nodulation in Medicago truncatula. Further, we demonstrate that the effect of light on nodulation may by mediated by the plant hormone ethylene. We propose to further characterize this regulatory network composed of light signaling and the ethylene pathway. Our goals are to determine how these two pathways interact, and how they function together to modulate nodulation. Ultimately, this research could result in recommendations for farmers of legume crops that could improve nodulation.

Animal Health Component

5%

Research Effort Categories

Basic

95%

Applied

5%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	1644	1050	25%
203	1644	1080	50%
206	1644	1050	25%

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

Subject Of Investigation
1644 - Winter annual legumes;

Field Of Science
1080 - Genetics; 1050 - Developmental biology;

Keywords

medicago truncatula

rhizobium

nodulation

light signaling

ethylene

shade

Goals / Objectives
OBJECTIVE 1: DETERMINE THE POINT AT WHICH LIGHT SIGNALING INHIBITS NODULATION BY EXAMINING EXPRESSION OF EARLY RHIZOBIUM-RESPONSIVE GENES. Plants exposed to FR or Blue light form few to no nodules. Examination of the roots by eye reveals no small primordia. At what point is this process blocked Subtle morphological changes in the first few hours after inoculation with rhizobia are marked by dramatic changes in gene expression. We will follow expression of early nodulation genes using quantitative Reverse Transcriptase-PCR (qRT-PCR). OBJECTIVE 2: DETERMINE THE ROLE OF THE GASEOUS HORMONE ETHYLENE IN MEDIATING THE FR AND BLUE INHIBITORY SIGNALS. It seems likely that a hormone must function downstream of FR and Blue light to mediate the nodulation response of the root. Could it be ethylene Our preliminary results suggest that the ethylene-insensitive sickle mutants are resistant to the inhibitory effects of FR and Blue light as well. If so, this provides evidence for a role for the hormone ethylene in mediating the light response with respect to nodulation. We will repeat these preliminary results and further examine the effect of Red light on nodulation in sickle mutants. If the effects of Red light and ethylene are not additive, we will conclude that these two likely function in a single pathway. Finally, we will test our model that FR and Blue light inhibit nodulation by increasing ethylene biosynthesis by directly measuring ethylene production in FR or Blue-light treated plants. This work will be done in collaboration with Alison Fisher (Norwich University). OBJECTIVE 3. HOW DOES LIGHT SIGNALING MODULATE THE EFFECT OF JASMONIC ACID ON NODULATION Our preliminary data indicate that R/FR light may modulate the effect of JA on nodulation in a manner similar to the effect we found with ethylene. Could this be mediated by ethylene biosynthesis We propose to follow up on our preliminary data and to further examine the effect of Red light on ethylene biosynthesis in the presence of JA. If the stimulatory effect of Red light on nodulation is due to decreased production of ethylene, then increasing ethylene synthesis by adding the ethylene precursor ACC should decrease nodulation even in Red light.

Project Methods
Nodulation Assays: 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 plants grown for ethylene measurements will be grown on agar slants in disposable test tubes as described. Where needed, Jasmonic Acid (Sigma, L 9283) and ACC (1-aminocyclopropane-1-carboxylic acid; Sigma) will be added to the medium after autoclaving to achieve a final concentration of 1 or 10 μM. Plates will be sealed with surgical tape and placed in a vertical position, so that the roots grow down the surface of the agar. Test tubes will be placed in racks with an opaque plastic lid. 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. Growth chamber conditions are 20 degrees C, 40% humidity, with a 16 hour day/8 hour night cycle. Ethylene Measurements: Plants will be grown on BNM agar slants in test tubes. At the time of measurement, tubes will be sealed with a cap containing a pierceable septum, plants will be grown for 24 hours, and 1 ml of the headspace will be removed with a Hamilton syringe inserted through the septum. The headspace will be transferred to a 2 ml vial sealed with a pierceable septum (1 ml of air will be removed from the vial prior to the addition of the 1ml of air from the plant tube). The vials will then be transported to Norwich University for analysis by Alison Fisher. Alison Fisher measures ethylene routinely for her research using gas chromatography with flame ionization detection.

Progress 10/01/08 to 09/30/11

Outputs
OUTPUTS: The purpose of this project is to determine the mechanism by which Red and Far red light regulate the formation of nitrogen-fixing nodules in the model legume, Medicago truncatula. Far Red light is the light that plants see a lot of when they are shaded by other plants. Thus, testing the response of plants to Far Red light is very similar to testing the effect of plant crowding. We will specifically test the hypothesis that Red/Far Red light regulate nodulation by altering production or response to the gaseous hormone, ethylene. We examined whether the giraffe mutant plants that we found have altered response to Far Red light, both for nodulation and for other aspects of plant growth (such as shoot elongation and flowering time). We have found that giraffe plants are insensitive to Far Red light for inhibition of nodulation, indicating that the wild-type function of the GIRAFFE gene is required for the Far Red light response. We also found that providing giraffe plants with more ethylene (by adding the precursor molecule, ACC) can reduce nodule number, showing that giraffe plants can respond normally to ethylene. We did grafting experiments that determined that the GIRAFFE gene is required in the shoot, where much of the light is perceived, rather than in the root, where nodulation occurs. We have also tested another, more distantly related legume, Lotus japonicus, and found that Far Red light regulates nodulation in this plant species in the same way as it does in wild-type Medicago. This year, we examined the effect of different wavelengths of light on the expression of hormone biosynthetic enzymes and on hormone-responsive genes. PARTICIPANTS: Yucan Zhang is a graduate student who worked on this project. She participated in design of the experiments and developed skills in new areas to do this work. Ellen Slade (undergraduate) tested different wild accessions of Medicago truncatula to see if they had altered nodulation responses to increased Far Red light. She is currently examining the effect of light on the rhizobial symbiont nodulation gene expression. Alex Miller (undergraduate) assisted with genotyping putative giraffe double mutant plants, learning genetic and molecular techniques. Beck Powers (undergraduate) assisted with cosegregation analysis of the giraffe phenotypes, learning genetics and nodulation assays. TARGET AUDIENCES: Two students, one graduate and one undergraduate, have learned from participating in this project. For all three undergraduates, this was their first experience working in a lab, and it has given these young women a sense of what a career as a scientist could be like. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have found that this effect of Far Red light on nodulation occurs in at least two different legume species. Our data so far are consistent with Red light stimulating nodulation and Far Red light inhibiting nodulation by inducing production or response to the stress hormone, ethylene. Interestingly, we have also found that Red light functions by inhibiting enzymes that synthesize another hormone, abscisic acid, and stimulating enzymes that promote its destruction. Since Far Red light is what plants see when they are shaded by other plants, this effect is significant and could have an impact on our agricultural practices. Thus the results of this work investigating the role of light signaling on nodulation could have a direct impact on farmers, particularly those involved in more sustainable agricultural practices involving legumes and inoculants. Red and Far Red light are used by plants to sense shading and proximity to neighboring plants. Thus, our data may result in recommendations that increased attention be paid to plant spacing to regulate irradiation of neighboring plants by far red light when increased nodulation is desired.

Publications

• No publications reported this period

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: The purpose of this project is to determine the mechanism by which Red and Far red light regulate the formation of nitrogen-fixing nodules in the model legume, Medicago truncatula. Far Red light is the light that plants see a lot of when they are shaded by other plants. Thus, testing the response of plants to Far Red light is very similar to testing the effect of plant crowding. We will specifically test the hypothesis that Red/Far Red light regulate nodulation by altering production or response to the gaseous hormone, ethylene. This year, we have begun to test the whether the giraffe mutant plants that we found have altered response to Far Red light, both for nodulation and for other aspects of plant growth (such as shoot elongation and flowering time). We have found that giraffe plants are insensitive to Far Red light for inhibition of nodulation, indicating that the wild-type function of the GIRAFFE gene is required for the Far Red light response. We also found that providing giraffe plants with more ethylene (by adding the precursor molecule, ACC) can reduce nodule number, showing that giraffe plants can respond normally to ethylene. We are currently testing whether the GIRAFFE gene is required in the shoot, where much of the light is perceived, or in the root, where nodulation occurs. We have also tested another, more distantly related legume, Lotus japonicus, and found that Far Red light regulates nodulation in this plant species in the same way as it does in wild-type Medicago. Next year, we will continue these studies and examine levels of the hormone ethylene itself in the Medicago system, or measure levels of ethylene biosynthetic enzymes, to see how changing the light quality can change the level of this hormone. PARTICIPANTS: Yucan Zhang is a graduate student who worked on this project. She participated in design of the experiments and developed skills in new areas to do this work. Ellen Slade is an undergraduate who worked on this project. She is testing different wild accessions of Medicago truncatula to see if they have altered nodulation responses to increased Far Red light. TARGET AUDIENCES: Two students, one graduate and one undergraduate, have learned from participating in this project. This is Ellen's first experience working in a lab, and it has given her a sense of what a career as a scientist could be like. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
We have found that this effect of Far Red light on nodulation occurs in at least two different legume species. Our data so far are consistent with Far Red light inducing production or response to the stress hormone, ethylene. Since Far Red light is what plants see when they are shaded by other plants, this effect is significant and could have an impact on our agricultural practices. Thus the results of this work investigating the role of light signaling on nodulation could have a direct impact on farmers, particularly those involved in more sustainable agricultural practices involving legumes and inoculants. Red and Far Red light are used by plants to sense shading and proximity to neighboring plants. Thus, our data may result in recommendations that increased attention be paid to plant spacing to regulate irradiation of neighboring plants by far red light when increased nodulation is desired.

Publications

• No publications reported this period

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The purpose of this project is to determine the mechanism by which Red and Far red light regulate the formation of nitrogen-fixing nodules in the model legume, Medicago truncatula. We will specifically test the hypothesis that Red/Far Red light regulate nodulation by altering production or response to the gaseous hormone, ethylene. During this year we have set up a light system for White + Far red light and shown that it does alter the plants ability to make legume nodules. We have begun to assay ethylene production and have optimized to the point that we can detect ethylene in pooled roots from young plants. Now that the system is largely set up, we will begin to test our hypotheses over the next year. PARTICIPANTS: Yucan Zhang is a graduate student who worked on this project. She participated in design of the experiments and developed skills in new areas to do this work. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
At this point we are still setting up the system and have no major findings. The results of this work investigating the role of light signaling on nodulation could have a direct impact on farmers, particularly those involved in more sustainable agricultural practices involving legumes and inoculants. Red and Far Red light are used by plants to sense shading and proximity to neighboring plants. Thus, our data may result in recommendations that increased attention be paid to plant spacing to regulate irradiation of neighboring plants by far red light when increased nodulation is desired.

Publications

• No publications reported this period