CHARACTERIZATION OF THE EFFECT OF SALT STRESS ON NODULATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1004630
• Project No.: VT-H02104
• Project Start Date: Oct 23, 2014
• Project End Date: Sep 30, 2019

Project Director
Harris, J.

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

Performing Department
Plant Biology

Non Technical Summary
The ability of legumes to form a symbiosis with nitrogen-fixing bacteria provides them with an internal source of fertilizer and is a lynchpin of sustainable agriculture. Nitrogen-fixing legume root nodules are the result of a symbiosis between legumes and soil bacteria collectively known as Rhizobium: only when the two symbiotic partners function together, can they fix nitrogen. The process of nodulation is an agriculturally important interaction, because not only are legumes productive without added nitrogen fertilizer, but they can also improve the nitrogen nutrition of plants grown nearby. The process of nodulation is sensitive to salt stress and is inhibited even at levels that do not have a large effect on plant biomass. Increased temperature, due to changes in overall climate, is leading to periodic drought stress, which results in the accumulation of salts in the soil as water evaporates. This increased salt in agricultural soils inhibits legume nodulation. We are studying the effect of salt stress on nodulation in the model legume, Medicago truncatula, which is closely related to the common forage crop, alfalfa (Medicago sativa).Although exposure to high salt can be stressful to plants, some studies show that pretreating plants with moderate concentrations of salt, called "salt acclimation", can reduce the negative effects of a subsequent high salt treatment on plant growth. Might M. truncatula plants also respond to salt acclimation by reducing detrimental responses when shifted to salt stress conditions? Would salt acclimation help protect M. truncatula nodulation against the stress of high salt? What is the mechanism of this response? We propose to test these questions in this project by examining the effect of salt acclimation and salt stress on nodulation by monitoring plant gene expression, cell division and Rhizobial infection. We will also examine the natural diversity of M. truncatula and ask whether certain genetic signatures correlate with salt resistance. We hope that together, these approaches will help us to better understand the effect of salt stress on nodulation and determine whether salt acclimation can ameliorate the negative effects of salt. In addition, we hope to identify some salt-tolerant or salt-acclimation associated genes, that may be useful to plant breeders selecting salt-resistant strains of crop legumes, such as alfalfa.

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
206	1644	1020	50%
206	1644	1080	50%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1644 - Winter annual legumes;

Field Of Science
1080 - Genetics; 1020 - Physiology;

Keywords

salt stress

nodulation

medicago truncatula

heme oxygenase

Goals / Objectives
The goals of this project are to determine how salt stress affects legume nodulation and whether gradual acclimation to salt can help mitigate some of the negative effects of high salt on nodulation.Objectives are:1. Determine how salt signaling intersects with early nodulation signaling in plant roots.We know a lot about the early steps in establishing this symbiosis in the plant. We also know that salt stress reduces nodule numbers, so it must block nodulation at a fairly early step. Where do these two pathways intersect?2. Test whether gradual acclimation of plants to salt will reduce the inhibitory effect of high salt on nodulation.Salt acclimation has been shown to promote biomass accumulation under high salt conditions that would otherwise be inhibitory to plant growth. Might it also work to restore symbiotic nodulation under high salt conditions? We will test this hypothesis both in the model legume Medicago truncatula and the crop legume, garden pea.3. Identify nodulation-associated genes whose expression is altered by salt stress or salt acclimation, using RNA-seq technology.We will take an unbiased, high-throughput approach to examine the entire Medicago genome to identify plant nodulation genes whose expression is altered by salt stress, and see whether a period of salt acclimation can promote a return to normal levels of gene expression.4. Test whether the GIRAFFE Heme Oxygenase enzyme plays a role in salt acclimation and salt tolerance in nodulation.The Heme Oxygenase enzyme is involved in salt acclimation and tolerance in Arabidopsis seed germination and early seedling growth. Is it also required for salt tolerance of legume nodulation?

Project Methods
This project will evaluate the effect of salt stress and salt acclimation on nodulation and on the very early steps of nodule development. Plants will be grown and inoculated in growth pouches, which allow us to access and the roots and visualize root and nodule development. We will compare the effect of different salt treatments on nodulation. Nodules will be counted visually, 14 days after inoculation with Rhizobium bacteria. We will monitor cell division using the fluorescent dye DAPI, which labels chromosomes. Infection will be followed using Rhizobium strains constitutively expressing either GFP or lacZ to follow the process of infection of M. truncatula roots using light or fluorescence microscopy. Gene expression will be evaluated using either a stably integrated reporter gene fusion, ENOD11-GUS, or high-throughput RNA-seq analysis. Specific genes may be additionally tested for differences in expression under different treatment conditions using quantitativeRT-PCR. To test for phenotypic variation for salt sensitivity in Medicago truncatula's natural range, we propose to query the Medicago HapMap database for single nucleotide polymorphisms (SNPs) in the GIRAFFE gene. Using this SNP information, we will identify unique haplotypes at GIRAFFE and test whether these haplotypes are correlated with decreased precipitation (and thus increased salinity) using using a mixed-linear effects model that accounts for population structure.

Progress 10/23/14 to 09/30/19

Outputs
Target Audience:The target audience includes scientists interested in salt stress as well as those interested in legume nodulation. Ultimately, plant breeders will be an audience if we are able to identify possible target genes for plant breeding. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This has provided an opportunity for graduate student Sanhita Chakraborty to develop expertise in large scale transcriptome analysis. In addition, an undergraduate student, Matthew Gorstein, used confocal microscopy to examine the effect of a moderate salt stress on early cell divisions during nodule development. Matthew developed his skills to a high level. He was offered an internship at the North Carolina Botanical Garden, but that was cancelled due to Covid. Sanhita is currently in a postdoctoral position at the University of Wisconsin. How have the results been disseminated to communities of interest?Results were presented as oral presentations at the University of Vermont and were to be presented at the North American Nitrogen Fixation conference in 2020. In addition, a manuscript is under review. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Previously, we determined that a moderate salt stress (100 mM NaCl) inhibits nodulation, reducing both infection of symbiotic Rhizobium and their differentiation into a nitrogen-fixing form. This past year we analyzed the results of our RNAseq approach to identify the set of genes induced by salt that are returned to normal levels by Rhizobium inoculation. We also examined the effect of this moderate salt stress on nodule development, using confocal microscopy to examine the earliest stages in nodule development. We found that the later stages of development were underrepresented, suggesting a block or delay in development by salt stress. Additionally, we found a number of developing nodules that had an abnormal pattern of cell divisions, suggesting that the nodules formed from these might be defective.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience includes scientists interested in salt stress as well as those interested in legume nodulation. Ultimately, plant breeders will be an audience if we are able to identify possible target genes for plant breeding. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This has provided an opportunity for graduate student Sanhita Chakraborty to develop expertise in large scale transcriptome analysis. In addition, an undergraduate student, Emily Millar, determined the quantity of the hormone Abscisic Acid (ABA) in symbiotic nodules grown in control or salt-stressed conditions. She also imaged infected nodules to determine whether salt affects infection of the symbiont into nodule cells. Emily developed her skills to a high enough level that she was hired as a research technician for a USDA lab. Sanhita has accepted a postdoctoral position at the University of Wisconsin. How have the results been disseminated to communities of interest?Results were presented as oral presentations at two International research conferences. In addition, Sanhita gave an oral presentation at our University-wide student research conference. Emily presented her work as a poster at a national research conference. What do you plan to do during the next reporting period to accomplish the goals?We are completeing our analysis of the transcriptome profiling RNAseq data on roots treated with or without salt stress and with or without Rhizobium inoculation. This is being done in collaboration with the University of Minnesota. We are also completing our characterization of nodule development in salt-treated Medicago truncatula roots. To do this, we are using microscopic approaches to look at cell divisions in early nodule development. We are also checking gene expression associated with different stages of differentiation, both of the plant and the bacterial symbiont. We are also preparing a paper for publication

Impacts
What was accomplished under these goals? Previously, we determined that a moderate salt stress (100 mM NaCl) inhibits nodulation, reducing both infection of symbiotic Rhizobium and their differentiation into a nitrogen-fixing form. This past year we analyzed the results of our RNAseq approach to identify other genes responsive that are differentially expressed in response to salt and rhizobium at 1day post inoculation. We have found a number of genes that are co-regulated with ENOD11, which will be interesting to examine further. We have also found a subset of genes that are expressed only in the presence of salt AND rhizobium. This is an interesting set of genes that we hope will help us to understand the complex interaction of the response to this abiotic stress and symbiotic nodule formation. We found that some of these genes have been previously determined to be involved in infection of the plant root. We also found, that some of these genes have been identified as being involved in local adaptation to drier climate conditions, which are likely to have increased salinity, verifying the usefulness of our approach.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Keller, C., Maeda, J., Jayaraman, D., Chakraborty, C., Sussman, M. R., Harris, J.M., Ane, J-M., and L. Li (2018). Comparison of Vacuum MALDI and AP-MALDI Platforms for the Mass Spectrometry Imaging of Metabolites Involved in Salt Stress in Medicago truncatula. Frontiers in Plant Science 9: 1238

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience includes scientists interested in salt stress as well as those interested in legume nodulation. Ultimately, plant breeders will be an audience if we are able to identify possible target genes for plant breeding.The target audience includes scientists interested in salt stress as well as those interested in legume nodulation. Ultimately, plant breeders will be an audience if we are able to identify possible target genes for plant breeding. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This has provided an opportunity for graduate student Sanhita Chakraborty to develop expertise in the effects of salt stress on plant growth and to develop molecular skills needed to examine gene expression and do high throughput transcriptome analysis. This year, Sanhita has begun to analyze her results from the transcriptome analysis and is learning how to think about big data sets. In addition, an undergraduate student, Emily Millar, has joined the project and is doing her honors thesis determining the quantity of the hormone Abscisic Acid (ABA) in symbiotic nodules grown in control or salt-stressed conditions. Emily has learned how to extract ABA and is learning how to measure the amount using an ELISA assay. She is also imaging infected nodules to determine whether salt affects infection of the symbiont into nodule cells. I mentored Emily in grant-writing and she received a college-level grant to fund her research one semester this year. A volunteer, Ashley Lantigua, worked on this project part-time for almost a year. She developed her skills to the point that she was able to get a technician job at UC Berkeley. How have the results been disseminated to communities of interest?Results were presented at a National research conference, and Sanhita's abstract was chosen for a talk. In addition, Sanhita gave an oral presentation at our University-wide student research conference. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete our analysis of the transcriptome profiling RNAseq data on roots treated with or without salt stress and with or without Rhizobium inoculation. This is being done in collaboration with the University of Minnesota. We plan to complete our characterization of nodule development in salt-treated Medicago truncatula roots. To do this, we are using microscopic approaches to look at cell divisions in early nodule development. We are also checking gene expression associated with different stages of differentiation, both of the plant and the bacterial symbiont. In addition, we will complete our measurements of Abscisic acid levels in symbiotic nodules, with or without salt treatment. Finally, we will begin to test giraffe mutant plants for salt sensitivity and expression of the ENOD11 gene. We are also preparing a paper for publication

Impacts
What was accomplished under these goals? Previously, we determined that a moderate salt stress (100 mM NaCl) inhibits nodulation, reducing both infection of symbiotic Rhizobium and their differentiation into a nitrogen-fixing form. This past year we looked more closely at the effect of a moderate salt stress on the early stages of the symbiosis, and found that salt has a dynamic effect on expression of the ENOD11 gene, commonly used as a measure of activation of the nodulation pathway, and that this requires the function of the NSP2 transcription factor. We also found that the effect of NaCl is due to an ionic effect, since KCl can mimic the effect of NaCl, but not drought or osmotic stress. In addition, we showed that the bacterial nitrogen-fixation genes are not turned on in plants subjected to a salt stress for 4 days prior to inoculation, but are turned on in plants that have only seen salt for 1 day prior to inoculation. Finally, we found that production of the bacterial exopolysaccharide, succinoglycan, modulates the response of ENOD11 to salt stress. This is an interesting result, because succinoglycan is absolutely required for bacterial infection, and a defect in bacterial infection is one of the strongest effects of salt stress that we see. We have also begun to analyze the results of our RNAseq approach to identify other genes responsive that are differentially expressed in response to salt and rhizobium at 1day post inoculation. We have found a number of genes that are co-regulated with ENOD11, which will be interesting to examine further. We have also found a subset of genes that are expressed only in the presence of salt AND rhizobium. This is an interesting set of genes that we hope will help us to understand the complex interaction of the response to this abiotic stress and symbiotic nodule formation.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Harris, Jeanne M. and Ondzighi-Assoume, Christine A. (2017). Environmental Nitrate Signals Through Abscisic Acid in the Root Tip. Plant Signaling and Behavior 12: 1, e1273303. http://dx.doi.org/10.1080/15592324.2016.1273303

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience includes scientists interested in salt stress as well as those interested in legume nodulation. Ultimately, plant breeders will be an audience if we are able to identify possible target genes for plant breeding. Changes/Problems:We found that salt acclimation did not significantly reduce the negative effect of salt on legume nodulation. As a result, moving forward our work will concentrate on our other goal of examining the direct effect of salt stress on nodulation, expanding our analysis to include quantification of the salt-responsive hormone, abscisic acid, in roots and shoots of our salt-stressed, inoculated plants. What opportunities for training and professional development has the project provided?This has provided an opportunity for graduate student Sanhita Chakraborty to develop expertise in the effects of salt stress on plant growth and to develop molecular skills needed to examine gene expression and do high throughput transcriptome analysis. Sanhita was selected to give an oral presentation on her research at a national meeting last summer. In addition, an undergraduate student, Emma Denman, joined the project and has developed the greenhouse pea nodulation assay. She has not only learned good experimental design, she has also learned some project management as she had to coordinate lab members helping out on this project. I mentored Emma in grant-writing and she received a college-level grant to fund her research one semester this year. How have the results been disseminated to communities of interest?Results were presented at a National research conference. In addition, Sanhita gave an oral presentation at our University-wide student research conference. What do you plan to do during the next reporting period to accomplish the goals?We plan to analyze the transcriptome profiling data we are running on roots treated with or without salt stress and with or without Rhizobium inoculation. We plan to complete our characterization of infection and nodule development in salt-treated Medicago truncatula roots. In addition, we plan to begin to measure levels of Abscisic acid in our inoculated roots, with or without salt treatment. Finally, we will begin to test giraffe mutant plants, as well as different M. truncatula accessions containing unique GIRAFFE haplotypes for salt sensitivity and expression of the ENOD11 gene, which we have shown is associated with the response of nodulation to salt stress.

Impacts
What was accomplished under these goals? We have determined that a moderate salt stress (100 mM NaCl) inhibits nodulation, reduces both infection of symbiotic Rhizobium and their differentiation into a nitrogen-fixing form. In addition, we tested expression of 6 known early nodulation genes. We found that, although this salt stress strongly inhibits nodulation (by 95%) and infection, in fact it stimulates expression of the Rhizobium-induced ENOD11 gene and one of its transcriptional regulators, ERN1, but has no effect on the early expression of other early nodulation genes. We identified 13 distinct haplotypes of the GIRAFFE heme oxygenase gene in a set of 292 different M. truncatula accessions. We have developed conditions for examining the effects of moderate salt stress on nodulation of garden pea, and found that it inhibits the maturation of the nodules.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ondzighi-Assoume, C., Chakraborty, S. and Jeanne Marie Harris (2016). Environmental Nitrate Stimulates Root Tip Abscisic Acid Accumulation via Release from Inactive Stores. Plant Cell 28(3):729-45.

Progress 10/23/14 to 09/30/15

Outputs
Target Audience:the target audience here was other scientists. The review article I published was in an Open Access journal, so that it it is accessible to all interested readers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This has provided an opportunity for graduate student Sanhita Chakraborty to develop expertise in the effects of salt stress on plant growth and to develop molecular skills needed to examine gene expression and do high throughput transcriptome analysis. Sanhita presented her work on this project at a scientific conference in the summer. In addition, an undergraduate student, Emma Denman, has joined the project and is learning how to analyze nodulation in pea plants. She is setting up conditions that will allow us to test the effect of salt stress on pea nodulation. How have the results been disseminated to communities of interest?Results were presented at a National research conference. In addition, a review article on the effects of abscisic acid on root architecture was written for a scientific audience. What do you plan to do during the next reporting period to accomplish the goals?We plan to determine the best time to do the transcriptome profiling on acclimated roots and send the RNA to our collaborators in Minnesota. We also plan to continue our characterization of infection and nodule development in salt-treated Medicago truncatula roots. In addition, we plan to begin to test the pea heme oxygenase mutant in our salt stress assay to see whether it is more, or less, sensitive to salt stress for nodulation.

Impacts
What was accomplished under these goals? We have learned that salt does not affect the initial infection steps by the symbiotic bacteria, nor the earliest changes in gene expression that we have assayed. However, by 3 days, we start to see changes in gene expression in salt-treated plants. We find that salt treatment lowers the levels of nodulation genes associated with nodule development. We are using this analysis to determine the best time point for our RNAseq analysis, to be done later this year. We are testing different salt acclimation treatments to find conditions that reduce the negative effects of salt on nodulation. We have found that a pretreatment with 25 mM NaCl reduces the overall level of nodulation, but increases the percent of mature root nodules. We are continuing to pursue this observation to see if we can optimize further.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Harris, Jeanne Marie. (2015). Abscisic Acid: Hidden Architect of Root System Structure. Plants, 4:548-572; doi:10.3390/plants4030548.