Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to NRP
IDENTIFICATION AND MANIPULATION OF REGULATORY MECHANISMS TO ENHANCE SUBMERGENCE AND DROUGHT TOLERANCE IN SOYBEAN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1001192
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2013
Project End Date
Sep 30, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Crop & Soil Environmental Sciences
Non Technical Summary
Flooding and drought are major damaging constraints to soybean production in the U.S. In Missouri, Arkansas, and Mississippi, approximately 50% of the soybean acreage is affected by flooding annually. US soybean production is also largely influenced by drought since more than 85% of soybean is produced by rain-fed farming. In Virginia, soybean is a major field crop (580,000 acres in 2012), but its productivity has been negatively affected by various environmental stresses including drought and flooding. Despite the economic impact of water-related stress in the major legume, fundamental mechanisms that regulate submergence and drought tolerance are largely unknown, presumably because the entire genome sequence and molecular tools were not available until recently. As a postdoctoral researcher, Takeshi Fukao led the USDA-NIFA-funded Sub1 Rice Project (2003-2012) exploring the mechanisms of submergence and drought tolerance in rice (Oryza sativa L.) with Prof. Julia Bailey-Serres at UC Riverside. Fukao has successfully adapted molecular protocols established in Arabidopsis for rice research and characterized the master regulator of submergence tolerance, SUBMERGENCE-1A (SUB1A), at physiological, molecular, and genomic levels (Fukao et al., 2006; Fukao and Bailey-Serres, 2008; Fukao et al., 2009; Fukao et al., 2011; Fukao et al., 2012). In this project, his advanced understanding of stress survival strategies in rice and Arabidopsis will be applied to increase the adaptability of soybean to extremes in precipitation under changing climate conditions. His ultimate goal is systematic elucidation of the intricate regulatory networks that govern adaptation and tolerance to an excess and insufficiency of water in the major legume species, aiding to enhance submergence and drought tolerance in commercially important soybean varieties in Virginia and other states.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2031820104050%
2031820102050%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1820 - Soybean;

Field Of Science
1040 - Molecular biology; 1020 - Physiology;
Goals / Objectives
1) Evaluate the contribution of rice SUB1A and soybean Group VII ERF genes to submergence and drought tolerance in soybean. 2) Define and manipulate key physiological and molecular components that affect adaptation to submergence and reoxygenation in soybean at vegetative stage. 3) Evaluate the effect of seed carbohydrate composition on seedling establishment following submergence.
Project Methods
Objective 1: Evaluate the contribution of rice SUB1A and soybean Group VII ERF genes to submergence and drought tolerance in soybean. 1.1 Assessment of the functional importance of rice SUB1A gene in submergence and drought tolerance of soybean Soybean transgenic plants overexpressing rice SUB1A genewill be developed using Agrobacterium-mediated transformation Homozygous T2 transgenic lines and William 82 will be analyzed for stress tolerance evaluation. Key physiological and molecular responses associated with submergence and drought will be evaluated in stress-treated plants. Plants at V1 stage will be completely submerged for 0, 1, 3, or 5 days. For reoxygenation, submerged plants will be removed from water at each time point and placed under normal growth conditions for 1 day. For drought, plants will be exposed to water deficit for 0, 3, 4, or 5 days. The first trifoliolate leaves of stressed plants will be harvested and immediately frozen in liquid nitrogen. For seedling establishment, 3-d-old etiolated seedling will be submerged for 0, 1, 2, or 4 day in the dark. The levels of carbohydrates (starch, sucrose, fructose, and glucose), ATP, and ADP will be quantified by standard enzymatic methods. Transcript accumulation of representative genes associated with adaptation to submergence and drought will be monitored by quantitative RT-PCR. Such genes include alcohol dehydrogenases, sucrose synthases, α-amylases, DREB1s, and dehydrins (LEA). 1.2 Assessment of the functional importance of soybean ERF VII genes in submergence and drought tolerance of soybean To determine whether submergence or ethylene affects mRNA accumulation of soybean ERF VII, the level of each gene transcript will be monitored by quantitative RT-PCR in submerged or ethylene-treated tissue. Based on the expression level, timing, location, and phylogenetic analysis, we will select candidate ERF VII (2-3 genes) associated with the regulation of adaptation to submergence in soybean. Overexpression and RNAi lines of each candidate genewill be created using Agrobacterium-mediated transformation. The functional roles of validated ERF VII genes in submergence and drought responses will be evaluated as described in Objective 1.1 (e.g., metabolite assays, hormone biosynthesis and response analysis, and gene expression analysis). 1.3 Identification of the tolerant specific allele(s) of the validated ERF VII genes from highly tolerant varieties (Year 4-5) We will survey submergence and drought tolerance in diverse soybean varieties (~50 accessions) obtained from USDA Soybean Germplasm Collection. We will also re-analyze 18 Brazilian soybean accessions provided by Dr. Alexandre Lima Nepomuceno (Embrapa Soybean, Brazil). Based on the results of submergence and drought tolerance screening, representative tolerant, intermediate, and intolerant lines (~30 lines) will be selected and the coding region of each validate ERF VII gene will be sequenced in the selected lines. The information on tolerant-specific alleles and donor accessions will be provided to Dr. Bo Zhang (Soybean breeder, Department of Crop and Soil Environmental Sciences, Virginia Tech). Objective 2: Define and manipulate key physiological and molecular components that affect adaptation to submergence and reoxygenation in soybean at vegetative stage. 2.1 Evaluation of phenotypic diversity in response to submergence and reoxygenation in soybean at vegetative stage Common physiological and biochemical responses to submergence will be monitored in developmentally matched plants as described in Objective 1.1. The samples will also be subjected to gene expression analysis by qRT-PCR. Transcript accumulation of alcohol dehydrogenase, sucrose synthase, and submergence-inducible ERF VII identified in Objective 1.2 will be monitored as molecular markers. By evaluation of stress response over the time-course, we will determine appropriate duration of stress for the global gene expression studies in Objective 2.2. 2.2 Characterization of the molecular response to submergence and reoxygenation in soybean, and comparative analysis of genome-scale gene expression data from soybean, Arabidopsis, and rice RNAseq analysis will be performed to gain genome-level understanding of the molecular response to submergence in tolerant (Essex) and intolerant (V71-370) soybean varieties. William 82 will also be used as a reference line since its entire genome sequence is publicly available. Optimal duration of submergence stress and recovery will be determined based on the results from Objective 2.1. 2.3 Evaluation of the functional role of the selected signaling components in submergence and reoxygenation tolerance Based on the results of transcriptome profiling and cross-species comparison in Objective 2.2, we will select candidate genes that may regulate submergence tolerance in soybean. Such candidates (~5 genes) include genes encoding transcription factors, kinase, phosphatase, and other signaling components. The functional importance of these genes in submergence tolerance will be evaluated in transgenic soybean or Arabidopsis. Objective 3: Evaluate the effect of seed carbohydrate composition on seedling establishment following submergence. 3.1 Quantification of carbohydrate components in soybean seeds. The abundance of major carbohydrates in cotyledons and hypocotyls will be monitored in V71-370 (Glycine max) and PI407162 (Glycine soja) during submergence. Etiolated seedlings will be completely submergence in a beaker for up to 4 days at 25 oC in the dark. Cotyledons and hypocotyls will be harvested at day 0, 1, 2, 3, and 4 and immediately frozen in liquid nitrogen. Dry seeds are also subjected to carbohydrate assays. The level of starch will be assayed as described in Objective 1.1. The content of stachyose, raffinose, sucrose, fructose, and glucose will be quantified by high-performance liquid chromatography (HPLC). To further evaluate the effect of carbohydrate composition and management on submergence tolerance, we will analyze representative RILs (F10 generation) derived from a cross between V71-370 and PI407162. Based on the published phenotypic data of 308 RILs, we will select five lines that vary in seed sucrose content, but are similar in seed size. The representative lines will be subjected to submergence tolerance evaluation and carbohydrate assays as described above. To validate the functional significance of seed carbohydrate component(s) in submergence tolerance, submergence survival tests will be performed in the presence and absence of carbohydrate(s). 3.2 Evaluation of metabolic pathways associated with key carbohydrates contributing to seedling establishment following submergence at the transcription level Transcript accumulation of genes involved in biosynthesis and degradation pathways of validated carbohydrates will be monitored during submergence in V71-370, PI407162, and five RILs analyzed in Objective 3.1. 3.3 Mapping QTL affecting seedling establishment following submergence and seed carbohydrate composition A total of 308 RILs have been genotyped with over 1,000 SSR and SFP markers. Of these RILs, 200 lines will be randomly selected for submergence survival evaluation at seedling establishment and carbohydrate quantification in seeds. Seedling survival of submergence will be quantified as described in Objective 1.1.Carbohydrate contents will be assayed using our standard protocol. The phenotypic data will be analyzed together with over 1,000 available molecular markers on the corresponding RILs. For QTL analysis, interval mapping will be performed with MapQTL 6. 3.4. Integration of QTL data with soybean physical maps and expression data Identified QTL regions in Objective 3.3 above will be related to the Williams 82 whole genome shotgun (WGS) sequences. To narrow down the candidate genes, we will also integrate physical locations of these genes with expression profiles obtained by quantitative RT-PCR.

Progress 10/01/13 to 09/30/18

Outputs
Target Audience:PlantPhysiologists, Plant Breeders, Graduate and Undergraduate students who are interested in environmental stress responses and tolerance in plants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1) Over the project period, three Ph.D. students received multidisciplinary training in physiology, molecular biology, genomics, and computational biology, two of who completed their Ph.D. degree. Currently, they work as postdoctoral researchers at University of Minnesota and Texas A&M University, respectively. The third student completed his course works in May 2018, and is currently focusing on his research work. All students took training courses on general lab safety, genetically modified organisms, and conflict of interest. Also, they participated in multiple national conferences for professional development. 2) This project also provided four undergraduate students with opportunities to experience authentic research experiences. Two of them graduated at Virginia Tech; One is currently working as a Ph.D. student at Dartmouth College; the other is working as an Associate Scientist at Pharmaceutical Product Development, LLC. All students took training courses on general lab safety, genetically modified organisms, and conflict of interest. How have the results been disseminated to communities of interest?1) 7 peer-reviewed publications 2) 3 oral presentations at international conferences 3) 1 oral presentations at national /regional conferences 4) 5 poster presentations at national / regional conferences 5) 2 Ph.D. dissertations 6) The research outcomes have also been used as teaching materials for PI Fukao's undergraduate courses such as "Crop Physiology and Ecology" and "Plant Breeding and Genetics". What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact Crop productivity is largely influenced by their ability to adapt or tolerate to abiotic stresses. Global climate change affects the magnitude and frequency of hydrological fluctuations, causing increased flood and drought events in the United States and other countries. To satisfy food, feed, and bioenergy demands under changing climates, it is necessary to improve crops tolerance to flooding and drought in a timely manner. This project aims to advance our understanding of the molecular and physiological mechanisms underlying adaptation to precipitation extremes in soybean. During the project period, this aim was expanded to rice and wheat. The results generated from the project will enhance our ability to develop new crop accessions with enhanced tolerance to flooding and drought through conventional breeding and genetic engineering. Accomplishments 1) We generated gene expression profiles of all nine soybean ERF-VII genes in leaves and roots under flooding and drought conditions. We also determined ethylene-responsive ERF-VII genes in soybean. These data were helpful to estimate the ERF-VII genes responsible to acclimation responses to precipitation extremes. 2) We performed microarray analysis to determine genes and pathways responsive to submergence and reoxygenation in soybean. Comparative analysis of differentially regulated genes in shoots and roots of soybean and other plants defines conserved, organ-specific, and species-specific adjustments that enhance adaptability to submergence and reoxygenation through different metabolic pathways. We also performed RNA-Seq analysis to characterize genes and pathways differentially regulated under submergence, drought, and recovery from these stresses in leaves and roots of soybean. Using the same tissues, we performed hormone profiling using LC-MS/MS. This analysis determined common, stress-specific, recovery-specific, and organ-specific responses in terms of gene expression and hormone biosynthesis/degradation in soybean. The useful data collected will help develop new accessions with enhanced tolerance to both submergence and drought through genetic engineering. 3) Our study revealed that soybean seedlings primarily consume starch as an energy resource under submergence, whereas soluble carbohydrates are maintained even after 7 d of submergence. We also investigated the role of a rice ERF-VII, SUB1A, in carbohydrate maintenance during submergence and recovery after de-submergence. SUB1A contributes to the restriction of starch and total soluble carbohydrate breakdown under submergence. This gene was also involved in rapid recovery of carbohydrate reserves in leaves after removal from water. SUB1A also played an important role in reduced carbohydrate degradation under prolonged darkness through the modification of hormonal pathways. These results enhanced our understanding of how the consumption of carbohydrate reserves are properly managed under stress and during recovery from stress conditions at the molecular and physiological levels. Avoidance of an energy crisis is key to enhance plant viability under flooded conditions, and a mechanistic understanding of carbohydrate management will facilitate to generate flooding-tolerant varieties using modern molecular breeding methods. 4) Additional accomplishment related to this project (1): The PI assisted data interpretation of evolutional analysis of rice ERF-VII genes in the project led by Antonio Oliveira at Universidade Federal de Pelotas, Brazil. Flooding tolerance of rice is controlled by the polygenic SUB1 locus containing two or three ERF-VII genes. This study revealed that the SUB1 locus exists on the same chromosomal location of most Oryza species with AA, BB, and FF genomes. The presence of SUB1 locus in Leersia perrieri, a flooding-tolerant grass, suggests that tandem duplication events creating SUB1A, B and C genes at this locus occurred before the speciation of this species. This study enhanced our knowledgeabout the evolutionary origin of this important submergence-tolerance gene in Genus Oryza. 5) Additional accomplishment related to this project (2): The PI contributed to gene expression analysis and data interpretation of physiological analysis in the project exploring the mechanism of submergence tolerance in wheat. This project was led by Ole Pedersen at University of Copenhagen, Denmark. This study revealed that the maintenance of leaf chlorophyll, leaf porosity, antioxidation capability is key for submergence tolerance in wheat. It appears that carbohydrate management under the stress is less important in wheat as compared to rice and soybean. These data emphasize that some mechanisms underlying plant tolerance to flooding are species-specific, and use of distinct approaches may be necessary to enhance flooding tolerance in wheat.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Herzog, M., Fukao, T., Winkel, A., Konnerup, D., Lamichhane, S., Alpuerto, J.B., Hasler-Sheetal, H., Pedersen, O. (2018) Physiology, gene expression and metabolome of two wheat cultivars with contrasting submergence tolerance. Plant, Cell and Environment 41: 1632-1644.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Fukao, T., Barrera-Figueroa, B.E., Juntawong, P., Pena-Castro, J.M. (2018) Submergence and waterlogging stress in plant: a review highlighting research opportunities and understudied aspects. Frontiers in Plant Science, Under review.
  • Type: Theses/Dissertations Status: Accepted Year Published: 2017 Citation: Jasper Alpuerto (2017) Physiological, metabolic, and transcriptional analysis of submergence tolerance in rice and nitrogen use efficiency in wheat. Ph.D. dissertation, Virginia Tech, Blacksburg.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Fukao, T. (2018) Waterproof rice gene, SUB1A  from genes to farmers fields. Egerton University, Njoro, Kenya. June 18, 2018. (Oral presentation by Takeshi Fukao).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Lamichhane, S., Alpuerto, J., Han, A., Fukao, T. (2018) Evaluation of the effect of prt6 mutation on tolerance to abiotic stresses in Arabidopsis and maize. Mid-Atlantic Section of American Society of Plant Biologists / University of Maryland Plant Symposium Joint meeting. College Park, MD. May 22-23, 2018. (Poster presentation by Suman Lamichhane).


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

Outputs
Target Audience:National and international researchers who are interested in environmental stress responses and tolerance in plants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1) Suman Lamichhane, a Ph.D. student, has performed computational analysis of our RNA-Seq data. He learned various statistical methods used for the interpretation of differentially expressed gene data. He mainly modified the existing software for soybean. Suman also improved his mentoring and leadership skills through the supervision of undergraduate research. 2) Another Ph.D. student, Jasper Alpuerto, has also be involved in this project. He prepared samples for hormone profiling. Jaspar also conducted expression analysis of individual genes by qRT-PCR. Jasper improved his mentoring and leadership skills through the supervision of undergraduate research. He passed his preliminary exam in April 2017. 3) Undergraduate researchers, Zachary Baker and Abbi Han, has assisted the two graduate students with plant culture, genotyping, and data analysis throughout the reporting period. 4) All lab workers completed training courses on general lab safety, genetically modified organisms, and conflict of interest in Virginia Tech. How have the results been disseminated to communities of interest?One peer-review paper on evolution of rice submergence tolerance locus was published in "Rice" in February 2017. Another peer-reviewed paper on multiple-level regulation of stress adaptation in plants was published in "Frontiers in Plant Science" in September 2017. The pdf files of these papers are available on the journal websites and ResearchGate. What do you plan to do during the next reporting period to accomplish the goals?We will complete data analysis of the RNA-Seq data (54 libraries) and submit a manuscript to a peer-reviewed journal. Specifically, we will connect our hormone profiling data with expression data of genes associated with hormone biosynthesis and degradation. This method will also be used for our amino acid profiling data. Collaborating with Dr. Song Li (Bioinformatics) at Virginia Tech, we will conduct gene network analysis to predict transcription factors that commonly regulates genes associated with flooding and drought responses.

Impacts
What was accomplished under these goals? Impact In the U.S., flooding and drought resulted in $4.4 and $4.1 billion losses in soybean production over the past 10 years, respectively. The overall goal of this project is to advance our understanding of how soybean adapts to water extremes at molecular and physiological levels. More specifically, we propose to identify key genes, biological processes, andhormones associated with acclimation responses to these stresses in soybean. The outcome of this project will aid in the development of new soybean varieties with increased tolerance to both flooding and drought. Accomplishments 1) We generated gene expression profiles of all nine soybean ERF-VII genes in leaves and roots under flooding and drought conditions. 2) We analyzed RNA-Seq data of leaves and roots of soybean exposed to flooding, drought, and recovery from these stresses (54 libraries). In this reporting period, we identified overrepresented gene ontology terms commonly or stress-specifically regulated under the stresses. Subsequently, we determined a subset of genes that represent in each GO category. This analysis recognized metabolic processes that are critical for both flooding and drought responses in leaves and roots, providinguseful information to develop new soybean accession with enhanced tolerance to both water extremes. We also performed hormone profiling in the tissues used for RNA-Seq. This analysis quantified over 20 compounds including hormones, their precursors and catabolites. Currently, we are analyzing the connection of these data with expression of genes associated with hormone biosynthesis and degradation. 3) This goal has been achieved in 2013-2014 and published in Tamang et al. (2014) Plant Cell and Environment. 4) Additional accomplishment related to this project: The PI assisted data interpretation of evolutional analysis of rice ERF-VII genes in the project led by Antonio Oliveira at Universidade Federal de Pelotas, Brazil. Flooding tolerance of rice is controlled by the polygenic SUB1 locus containing two or three ERF-VII genes. This study revealed that the SUB1 locus exists on the same chromosomal location of most Oryza species with AA, BB, and FF genomes. The presence of SUB1 locus in Leersia perrieri, a flooding-tolerant grass, suggests that tandem duplication events creating SUB1A, B and C genes at this locus occurred before the speciation of this species.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Haak, D., Fukao, T., Grene, R., Hua, Z., Ivanov, R., Perrella, G., Li, S. (2017) Multilevel regulation of abiotic stress responses in plants. Frontiers in Plant Science 8: 1564.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: dos Santos, R.S., Farias, D.R., Pegoraro, C., Rombaldi, C.V., Fukao, T., Wing, R., de Oliveira, A.C. (2017) Evolutionary analysis of the SUB1 locus across the Oryza genomes. Rice 10: 4.


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

Outputs
Target Audience:National and international researchers who are interested in environmental stress responses and tolerance in plants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1) This study was a part of the PhD research project for Bishal Tamang who started the graduate study in Fall 2013. Bishal completed all of his research works, submitted a dissertation, and received his PhD from Virginia Tech in August 2016. Through this project, Bishal received multidisciplinary training in physiology, molecular biology, genomics, and computational biology. He also attended weekly departmental seminars in Crop and Soil Environmental Sciences and presented his research results. Bishal improved his mentoring and leadership skills through supervision of undergraduate research. Currently, he is working as a postdoctoral researcher in University of Minnesota. 2) Another PhD student, Jasper Alpuerto, has also be involved in this project. His major responsibility is to develop transgenic Arabidopsis plants overexpressing soybean ERF-VII genes. In this project, Jasper learned the basic concept and techniques for plant genetics, transformation, and stress physiology. He also attended weekly departmental seminars in Crop and Soil Environmental Sciences and presented his research results. Jasper improved his mentoring and leadership skills through supervision of undergraduate research. 3) An undergraduate researcher, Peyton Scot, has assisted the two graduate students with plant culture, genotyping, and metabolite assays throughout the reporting period. She hopes to start her graduate study in Fall 2017 at another university. Her 2-year research experience obtained in this project willhelp to make her a competitive candidate for graduate school 4) All lab workers completed training courses on general lab safety, genetically modified organisms, and conflict of interest in Virginia Tech. How have the results been disseminated to communities of interest?1) One peer-reviewed paper on plant adaptation to submergence was published in "International Journal of Molecular Sciences" (IF: 3.257) in December 2015. The pdf file of this paper is available on the journal website and ResearchGate. 2) One PhD dissertation written by Bishal Tamang was submitted to Virginia Tech in August 2016. The pdf file of this article is available on the University website. 3) PD Fukao presented the research results as an invited speaker at the 12th Conference of the International Society for Plant Anaerobiosis in Elsinore, Denmark in September 2016. The pdf file of the abstract is available on the conference website. 4) A PhD student, Bishal Tamang, presented his poster at the 16th Biennial Conference of the Molecular and Cellular Biology of the Soybean in Columbus, OH in August 2016. The pdf file of the abstract is available on the conference website. What do you plan to do during the next reporting period to accomplish the goals?1) Arabidopsis overexpression lines will be used to evaluate tolerance to flooding and drought. The results will determine the functional role of ERF-VII genes in acclimation responses to the stress. 2) Computational analysis of the RNA-Seq data (54 libraries) will be completed. Such analyses include the identification of signaling and metabolic pathways involved in responses to submergence and drought and comparative analysis of the soybean data with publicly available Arabidopsis data. These results will determine stress-specific, tissue-specific, species-specific and overlapped pathways associated with submergence and drought tolerance.

Impacts
What was accomplished under these goals? Impact In the U.S., flooding and drought resulted in $4.2 and $4.5 billion losses in soybean production over the past 10 years, respectively. The overall goal of this project is to advance our understanding of how soybean adapts to water extremes at molecular and physiological levels. More specifically, we propose to identify key genes, signaling molecules, and metabolites associated with acclimation responses to these stresses in soybean. The outcome of this project will aid in the development of new soybean varieties with increased stress tolerance. Accomplishments 1) Overexpression of ERF-VII genes and stabilization of their proteins enhance flooding and drought tolerance in Arabidopsis, rice, and barley. Based on gene expression analysis performed in 2014-2015, we identified three soybean ERF-VII genes that are likely to be key regulators for adaptation to water extremes. In 2015-2016, we developed homozygous Arabidopsis transgenic lines overexpressing each of these three genes, which will be used for stress tolerance evaluation. 2) We conducted RNA-Sequencing to observe transcriptomic adjustments to submergence, drought, and recovery from these stresses in soybean leaves and roots in 2014-2015. In this reporting period, we conducted computational analysis of the sequencing data from 54 libraries. This analysis identified 15,075 gene transcripts that were significantly up- or down-regulated in response to submergence or drought in leaves or roots. We also conducted k-mean clustering analysis to classify differentially expressed genes into 30 types based on the expression patterns. Currently, we are analyzing overrepresented pathways (hormones, transcription factors, and other signaling components). Data analysis will be completed in the next reporting period. 3) This goal has been achieved in 2013-2014 and published in Tamang et al. (2014) Plant Cell and Environment.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Tamang, B.G., Fukao, T. (2015) Plant adaptation to multiple stresses during submergence and following desubmergence. International Journal of Molecular Sciences 16: 30164-30180.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Tamang, B.G., Rajasundaram, D., Li, S., Fukao, T. (2016) Crosstalk of transcriptomic responses to submergence and drought in leaves and roots of soybean. 16th Biennial Conference of the Molecular and Cellular Biology of the Soybean. Columbus, OH. August 7-10, 2016. (Poster presentation by Bishal Tamang).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Fukao, T., Tamang, B.G., Rajasundaram, D., Li, S. (2016) Comparative analysis of genome-scale gene expression responses to submergence, drought and recovery from these stresses in leaves and roots of soybean. 12th Conference of the International Society for Plant Anaerobiosis. Elsinore, Denmark. September 5-8, 2016. (Oral presentation by Takeshi Fukao).
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Tamang, B.G. (2016) Exploration of Physiological and Molecular Responses to Precipitation Extremes in Soybean and Nitrogen Fertility in Wheat. Ph.D. dissertation, Department of Crop and Soil Environmental Sciences, Virginia Tech.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:National and international researchers who are interested in environmental stress responses and tolerance in plants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A PhD student, Bishal Tamang, has largely conducted experiments in this project, receiving multidisciplinary training in physiology, molecular biology, genomics and computational biology. A postdoctoral scholar, Fraz Hussain initiated Arabidopsis transformation and a PhD student, Jasper Alpuerto, has grown these plants to get homozygous lines. An undergraduate student, Joseph Magliozzi, assisted the two PhD students and one postdoctoral scholar with plant culture and stress treatments. After graduation of Magliozzi in May 2015 (currently PhD student in Dartmouth College), an undergraduate researcher, Peyton Scot, was hired in June, 2015, currently learning basic lab techniques (buffer preparation, DNA extraction, genotyping, plant culture etc.). Postdoctoral, graduate and undergraduate researchers attended a weekly one-hour group meeting to present their research progress and recent papers published form other group. At the meeting, they received advice on public speaking and critical thinking skills. How have the results been disseminated to communities of interest?PI Fukao presented a talk on soybean submergence tolerance as an invited speaker at the New Phytologist Workshop on Flooding Stress in Ravenstein, the Netherlands on June 20, 2015. A PhD student, Bishal Tamang, presented his posters on soybean submergence tolerance at the Graduate Student Symposium, Department of Crop and Soil Environmental Sciences, Virginia Tech on February 6, 2015 and at the 20th Penn State Plant Biology Symposium on May 13-16, 2015. What do you plan to do during the next reporting period to accomplish the goals?1) Arabidopsis overexpression lines will be evaluated for functional analysis of selected soybean ERFVII genes. The results will determine the role of these genes in acclimation responses to submergence and drought. We will also test the function ofthese genes in stress tolerance using virus-induced gene silencing in soybean. 2) Transcriptional adjustments to submergence and drought in leaves and roots will be characterized by RNA-Seq technology. The results will be validated by individual gene expression analysis. The RNA-Seq date will be integrated with metabolite data, identifying key pathways associated with stress tolerance.

Impacts
What was accomplished under these goals? Submergence and drought are major natural disasters causing yield loss in soybean. In this project, we aim to advance our understanding of how soybean adapts to water extremes at molecular and physiological levels. More specifically, we propose to identify key genes, signaling molecules, and metabolites associated with acclimation responses to these stresses in soybean. The outcome of this project will aid in the development of new soybean varieties with increased stress tolerance. Accomplishments achieved in this project period. 1) It is known that ERF-VII transcription factors play a pivotal role in the regulation of submergence and drought tolerance in Arabidopsis, rice, and other species. To generate the expression profile of soybean ERF-VIIs, accumulation of their mRNAs were monitored in leaves and roots of soybean plants during submergence and drought. The effect of recovery from these stresses on expression of these genes was also observed. Based on the results, we selected two ERF-VII genes as key regulators for submergence and drought tolerance. To investigate their functional importance, we are developing Arabidopsis transgenic lines overexpressing each of these genes. Homozygous T3 seeds will be available in early 2016. We are also developing a protocol to verify the effect of these genes in soybean plants using virus-induced gene silencing. 2) We conducted RNA-Sequencing to observe transcriptomic adjustments to submergence, drought, and recovery from these stresses in soybean leaves and roots. Collaborating with Dr. Song Li, we are analyzing differentially expressed genes and alternatively spliced isoforms associated with stress adaptation and recovery. The data analysis will be completed in early 2016. Using the same tissues, we have monitored alternations in the abundance of carbohydrate and individual amino acids over the course of submergence and drought treatment. These data will be integrated with genome-scale gene expression data generated by RNA-Sequencing. 3) This goal has been achieved in 2013-2014 and published in Tamang et al. (2014) Plant Cell and Environment.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Tamang, B.G., Fukao, T. (2015) Differential regulation of metabolic responses to submergence in leaves and roots of soybean plants. The Graduate Student Symposium, Department of Crop and Soil Environmental Sciences, Virginia Tech. Blacksburg, VA, February 6, 2015 (Poster presentation by Bishal Tamang).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Tamang, B.G., Fukao, T. (2015) Tissue-specific responses of soybean plants to submergence stress and recovery at early vegetative stage. 20th Penn State Plant Biology Symposium, Plant Stress-Omics in a Changing Climate. University Park, PA, May 13-16, 2015 (Poster presentation by Bishal Tamang).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Fukao, T. (2015) Submergence responses in leaves and roots of soybean plants. New Phytologist Workshop on Flooding Stress. Ravenstein, the Netherlands, June 20-21, 2015 (Oral presentation by Takeshi Fukao).


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: National and international researchers who are interested in environmental stress responses and tolerance in plants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? PhD students, Bishal Tamang and Jasper Alpuerto, received multidisciplinary training in physiology, molecular biology, genomics and computational biology through the project. An undergraduate researcher, Joseph Magliozzi, assisted the two PhD students with plant culture and stress treatments. Graduate and undergraduate students attended a weekly 1.5-hour group meeting. The format includes a research presentation by one individual and a publication presentation by another individual. In addition, graduate students attended departmental seminar series in Fall and Spring semesters. The seminars are generally cross-disciplinary because the members in Department of Crop and Soil Environmental Sciences include molecular biologists, plant breeders, agronomists, soil scientists, environmental scientists, analytical chemists, and computational biologists. All lab workers including undergraduate researchers must complete training courses on general lab safety and conflict of interest in Virginia Tech. How have the results been disseminated to communities of interest? One research paper on soybean flooding responses was published in Plant, Cell & Environment in February 2014. One review papers on water-related stress in plants was published in Current Opinion in Plant Biology in October 2013. A PhD student, Bishal Tamang, presented his research results as an invited speaker at 15th Biennial Conference of Molecular & Cellular Biology of the Soybean in Minneapolis on August 4, 2014. An undergraduate researcher, Joseph Magliozzi, presented his research accomplishments at Virginia Tech Research Poster Symposium on May 1, 2014. Our microarray dataset were posted in Gene Expression Omnibus (GSE51710), which have been publicly available since February 18, 2014. What do you plan to do during the next reporting period to accomplish the goals? 1) Arabidopsis overexpression lines will be available to evaluate the functional role of the candidate ERF VII genes in submergence and drought tolerance. Using these genetic materials, we will characterize physiological and molecular acclimations to the stresses. The outcome will advance our understanding of the regulatory mechanism of ERF VII-mediated adaptations to submergence and drought. 2) Transcriptional adjustments to submergence in leaves and roots will be evaluated by RNA-Seq technology. The tissues used for RNA-Seq will also be subjected to hormone profiling. Transcriptome and hormone profiles will be integrated with the metabolite data obtained in this reporting period. This analysis will characterize conserved or tissue-specific transcriptional, hormonal and metabolic adjustments to submergence and reoxygenation in soybean. These results will be submitted to a refereed journal during the next reporting period. 3) We will quantify carbohydrate and phytate contents in seeds of mips1 mutants. Using near-isogenic lines of MIPS1 (MIPS1 vs. mips1), we will investigate the functional role of MIPS1 in submergence tolerance and seed sucrose/phytate contents. Analyses include comparative analysis of gene expression and carbohydrate degradation in seeds of near-isogenic lines during submergence.

Impacts
What was accomplished under these goals? 1) Our quantitative RT-PCR analysis identified three soybean Group VII ERF genes which are highly induced by submergence and ethylene. Phylogenetic analysis placed these three genes in the same clade, which is clearly separated from other soybean ERF VII genes. Currently, we are developing overexpression lines in Arabidopsis to analyze the function of the soybean genes in flooding and drought tolerance. 2) We monitored the abundance of stored carbohydrate and nitrogen compounds (nitrate, ammonium, individual amino acids, and proteins) in leaves and roots of soybean plants over the course of submergence and recovery. The same tissues will be subjected to genome-scale gene expression analysis by RNA-Seq and hormone profiling. These results will be submitted to a refereed journal in early 2015. 3) We surveyed three soybean lines containing a mutated myo-inositol phosphate synthase 1 (mips1) gene for submergence tolerance at the seedling establishment stage. Our stress tolerance evaluation demonstrated that the presence of mips1 positively correlates with submergence tolerance in young soybean seedlings. In general, soybean lines with mips1 contain low phytate and high sucrose in seeds. We will quantify sucrose and other carbohydrates as well as phytate in seeds. We will also investigate the functional role of mips1 in submergence tolerance and sucrose/phytate contents.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Fukao T., Xiong L. (2013) Genetic mechanisms conferring adaptation to submergence and drought in rice: simple or complex? Current Opinion in Plant Biology 16:196-204.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Tamang B.G., Magliozzi J.O.*, Maroof M.A.S., Fukao T. (2014) Physiological and transcriptomic characterization of submergence and reoxygenation responses in soybean seedlings. Plant, Cell & Environment 37: 2350-2365. * Undergraduate researcher.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Fukao T., Yeung E., Bailey-Serres J. (2013) SUB1A delays dark-induced senescence in rice. 11th Conference of the International Society for Plant Anaerobiosis. Los Banos, Philippines. October 6-11, 2013 (Oral presentation by T. Fukao).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Magliozzi J.O.*, Tamang B.G., Maroof M.A.S., Fukao T. (2014) Physiological and molecular characterization of submergence and reoxygenation responses in soybean seedlings. Virginia Tech Research Poster Symposium, Blacksburg VA, May 1, 2014. (Poster presentation by Joseph Magliozzi). * Undergraduate researcher.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Tamang B.G., Magliozzi J.O.*, Maroof M.A.S., Fukao T. (2014) Physiological and transcriptomic responses to submergence and reoxygenation in soybean at the seedling establishment stage. SOY2014: Molecular and Cellular Biology of the Soybean 15th Biennial Conference, Minneapolis, MN, August 3-6, 2014. (Oral presentation by Bishal Tamang). * Undergraduate researcher.