Progress 12/15/18 to 12/14/23
Outputs
Target Audience:Graduate, undergraduate, and high school students underwent training in molecular biology and epigenomic tools such as DNA and RNA isolation, library preparations, Polymerase Chain Reactions (PCRs), methylation assays, and chromatin immunoprecipitation assays. Additionally, students were involved in greenhouse and field experiments to conduct drought studies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Undergraduate, graduate and doctoral students received training in cutting-edge molecular techniques. These skills included DNA and RNA isolations, c-DNA library preparation, diverse types of polymerase chain reaction applications, chromatin immuno precipitations; as well as RNA library preparations. Moreover, involvement extended beyond the laboratory setting into conference participation such abstract submissions for poster or oral presentations - even mentoring peers formed part of their dynamic learning experience. The students presented their findings in conferences. The training of graduate students encompassed dissertation preparation, including the analysis of results through statistical software such as GraphPad Prism. Moreover, the students were actively engaged in manuscript writing and familiarized them with all steps associated with publishing their findings How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Field Study Six genotypes of common bean were used. The experiment was set up with four replications of randomized blocks. Each block contained four rows spaced 56 cm apart and 3.6 m long. Plants were grown under drip irrigation until flowering, receiving a total of 135.1 mm of water, including 101.6 mm from irrigation and 33.5 mm from natural precipitation. After at least 50% of the plants flowered, a terminal drought was imposed. No irrigation was provided during this period, which saw 16.3 mm of precipitation. After 2-3 weeks of drought stress at the R7 stage, 18 samples were collected from 3 sensitive and 3 tolerant genotypes across 3 replicates. ENT. LINE CODE PEDIGREE Market Class ORIGIN 1 NE36-20-1 SB-DT2 Pinto NE28-18-18-14 2 NE36-20-2 SB-DT3 Small red NE28-18-18-21 3 NE36-20-3 Matterhorn Great Northern NE8-19-97 4 NE36-20-4 Merlot Small red NE28-18-18-31 5 NE36-20-5 Stampede Pinto NE28-18-18-32 6 NE36-20-6 Sawtooth Great northern GH20 Results and Outcome a). Terminal Drought Impacts on Common Bean Root Gene Expression (Graduate Student Research) We analyzed the expression of three drought-responsive genes - sucrose synthase (SuS), lipid transfer protein (LTP), and 9-cis-epoxycarotenoid dioxygenase (NCED) - across six common bean genotypes grown under terminal drought and non-drought conditions. The findings revealed a significant decrease in SuS expression in drought-stressed plants, likely showing a metabolic shift to conserve resources when water is limited. Alternatively, LTP expression markedly increased under drought stress, suggesting it may play a role in stress tolerance mechanisms in common beans. Unlike the other two genes, NCED expression showed no major differences between treatments, causing further research to elucidate its precise function in the ABA-mediated drought response pathway. Overall, the distinct expression patterns of SuS and LTP point to specific roles for these genes in how common beans respond and adapt to drought stress through metabolic changes and activation of protective mechanisms. b). Terminal Drought Response in Common Bean: A Transcriptome Analysis Using comparative transcriptome analysis of roots and leaves, significant differences in differentially expressed genes (DEGs) between the two genotypes were observed. The sensitive genotype exhibited a higher proportion of downregulated DEGs, suggesting a hampered stress response. Several DEGs overlapped between genotypes and were present in a drought-specific database, indicating their importance in drought response mechanisms. Key gene families involved in stress response, protein folding, and cell protection were identified, including transcription factors, heat shock proteins, and chaperones. DEGs associated with abscisic acid (ABA)-regulated drought stress responses showed higher expression in both genotypes, highlighting the importance of this pathway. DEGs in the tolerant genotype were specifically involved in signal transduction, oxidative stress damage repair, and transportation processes, potentially explaining the observed temporal pattern of root growth and ABA-dependent drought tolerance. The KEGG pathway analysis showed that various pathways were interacting with each other, with transcription factors linked to ERF, BHLH, EIL, and bZIP pathways being particularly involved in enhancing drought tolerance in the genotype that can withstand drought. In essence, this research sheds light on how molecular processes contribute to plants' ability to endure drought and presents potential avenues for creating drought-resistant plant varieties c). Exploring Seed Metabolomics in Common Bean Genotypes under Terminal Drought Stress Drought, like many other environmental challenges, can alter how plants store metabolic compounds. Our study investigated the metabolic profiles of resilient common bean varieties (Matterhorn, SB-DT2, and SB-DT3) compared to their sensitive counterparts (Sawtooth, Merlot, and Stampede) using advanced technology called Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS). We grew these beans in real field conditions and exposed them to drought stress after flowering, known as terminal drought stress. The results were significant: UPLC-MS revealed significant differences in seed metabolic profiles between tolerant genotypes (Matterhorn, SB-DT2, and SB-DT3) and sensitive ones (Sawtooth and Merlot). Despite growing them in the same conditions and subjecting them to post-flowering drought stress, we observed distinct variations, reflecting their different resilience levels. To understand how genotype-specific metabolites respond to terminal drought stress, we compared resilient and sensitive genotypes within the same group. This comparison identified 26 potential metabolites and associated metabolic pathways, including monobactam biosynthesis, flavone and flavonol biosynthesis, pentose phosphate pathway, and others. Notably, Flavonoid Biosynthesis was also evident in our findings. Comparing specific genotype pairs under terminal drought stress, we found varying metabolic pathways' significance, with SB-DT2 showing more significant outcomes against Stampede than Matterhorn did against Sawtooth, and SB-DT3 outperforming Merlot. By exploring how tolerant and sensitive common bean varieties respond to terminal drought, our study contributes vital insights into seed metabolite profiles and their associated pathways, aiding our understanding of this complex process d). Proteomic Insights into Terminal Drought Response in Common Bean Seeds In response to terminal drought stress, this study has identified proteomic changes in the seeds of two common bean genotypes: SB-DT3 and SB-DT2, both tolerant; and Merlot as well as Stampede which are sensitive. Abundant differentially expressed proteins (DEPs) were found in the susceptible genotype compared to the tolerant line. We have identified DEPs associated with starch biosynthesis, protein-chromophore linkage, and photosynthesis in both genotypes; however, a few DEPs along with enriched biological pathways exhibited specific differences based on their respective genotypes. Uniquely, the tolerant genotypes demonstrated Differential Expressed Proteins (DEPs) linked to sugar metabolism and plant signaling; in contrast, more DEPs associated with plant-pathogen interaction, proteasome function and carbohydrate metabolism were observed within the sensitive genotypes. DEP involvement in chaperone and signal transduction also showed significant alterations between both genotype types. To summarize: our proteomic analysis uncovered a range of conserved yet genotype-specific DEPs ripe for targeting through selective breeding programs aimed at developing drought-tolerant common bean varieties. e). Terminal drought's Impact on Bean Root Microbiome (Graduate Student Research) Understanding from a prior transcriptomic study drove our investigation into the influence of stress on the Common bean genotype's root microbiome diversity. We isolated and sequenced bacteria from roots grown under both stress and control conditions, successfully identifying a diverse range of species and genera. The 16S rRNA sequences, presently deposited in NCBI, may help in understanding the role that a plant's root microbiome plays in drought stress tolerance, furthermore, paving the way for prospective strategies based on microbiomes to support crop resilience. Upon successful 16S rRNA sequence analysis, the phylogenetic tree was generated, enabling subsequent whole-genome sequencing of six diverse isolated bacteria. 16S ribosomal RNA gene, partial sequence submission to NCBI https://www.ncbi.nlm.nih.gov/nuccore/?term=OR610688:OR610694[accn] https://www.ncbi.nlm.nih.gov/nuccore/?term=OR807306:OR807317[accn]
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Subramani M, Urrea CA, Habib R, Bhide K, Thimmapuram J, Kalavacharla V. Comparative Transcriptome Analysis of Tolerant and Sensitive Genotypes of Common Bean (Phaseolus vulgaris L.) in Response to Terminal Drought Stress. Plants. 2023; 12(1):210. https://doi.org/10.3390/plants12010210
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Subramani M, Urrea CA, Tamatamu SR, Sripathi VR, Williams K, Chintapenta LK, Todd A, Ozbay G. Comprehensive Proteomic Analysis of Common Bean (Phaseolus vulgaris L.) Seeds Reveal Shared and Unique Proteins Involved in Terminal Drought Stress Response in Tolerant and Sensitive Genotypes. Biomolecules. 2024; 14(1):109. https://doi.org/10.3390/biom14010109
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
ELUCIDATING THE INFLUENCE OF DROUGHT ON ENDOPHYTIC BACTERIAL DIVERSITY IN ROOTS OF COMMON BEAN (PHASEOLUS VULGARIS L.) USING PCR-BASED TECHNIQUES
KRYSTAL WILLIAMS, Mayavan Subramani,Carlos A Urrea, Antonette Todd, Gulnihal Ozbay.
Professional Agricultural Workers Conference (Tuskegee, Alabama) - October 28-November 1, 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
ELUCIDATING THE INFLUENCE OF DROUGHT ON ENDOPHYTIC BACTERIAL DIVERSITY IN ROOTS OF COMMON BEAN (PHASEOLUS VULGARIS L.) USING PCR-BASED TECHNIQUES
KRYSTAL WILLIAMS, Mayavan Subramani,Carlos A Urrea, Antonette Todd, Gulnihal Ozbay.
First State Black Farmers Conference (Dover, DE) - November 8-9, 2023
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Elucidating the Influence of Drought on Endophytic Bacterial Diversity in Roots of Common Bean (Phaseolus vulgaris L.) Using PCR-based Techniques. K. Williams*, M. Subramani, A. Todd, and G. Ozbay,2024.
ARD Biennial Research Symposium (Nashville, Tennessee) - April 6-9, 2024
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2021
Citation:
Understanding the Effect of Terminal Drought on Gene Expression in the Root of Different Genotypes of Common Bean (Phaseolus vulgaris.L).
Delaware State University, Dover, DE.19901.
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Progress 12/15/20 to 12/14/21
Outputs
Target Audience:Graduate and undergraduate students were trained in designing a randomized block design to carry out a drought experiment in the field. Additionally, students were trained in molecular biology techniques to understand the variations in gene expression between drought and control plants.? Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Several levels of students (whether funded or not funded) received training in chromatin isolation, sonication, nuclei isolation, and RNA library preparation. In addition, students participated in a mentoring process to demonstrate basic molecular biology techniques. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Transcriptomics and metabolomics and proteomics data are currently being analyzed. Preparation of manuscripts based on transcriptomics, metabolomics, and proteomics data.
Impacts
What was accomplished under these goals?
Drought study - Controlled Environment (FAIRE- Seq) The FAIRE -Seq (Formaldehyde-Assisted Isolation of Regulatory Elements - Sequencing) approach was used to explore the nucleosome-free chromatin regions in common bean under drought and rewatering conditions. In this study, young leaves from drought, control, and rewatered plants were collected to extract chromatin free DNA. Additionally, the DNA library was prepared for paired-end sequencing with the illumina HiSeqTM 2500. Bioinformatics tools such as FastQC, Trimgalore, Bowtie2, DeepTools, epic2,ChIPseeker, PlantTFDB, ClusterProfiler and MEME suite were used to analyse and find out the differential peaks between the treatments. Field Study - Transcriptomics We were unable to conduct a field study in Delaware. We were successful in conducting drought experiments in Nebraska. Transcriptomics was conducted on young leaves and roots from the plants that were grown in drought and controlled conditions in Nebraska. The RNA library was prepared and sequenced with the illumina HiSeqTM 2500. Bioinformatics tools such as PlantTFDB, Drought database, KEGG pathway, FASTQC, Trimgalore, STAR indexed genome using STAR, HTSeq package, DESeq2, clusterprofiler, R Package was used to compare the differential gene expression between the Leaf tolerance vs Sensitive, Root tolerance vs Sensitive and Tolerance root vs Tolerance leaves, Sensitive root vs Sensitive leaves. Untargeted Metabolomics and Proteomics of Bean seeds Bean seeds collected from drought stressed and control plants in the field were used in the integrated metabolomics and proteomics study. LC MS/MS and label-free proteomics were used in these studies. Results FAIRE-Seq -FAIRE peaks were found to be enriched in promoter regions in all samples (>50%). A very distinct pattern of peaks was observed in drought samples, which contrasted with control and rewatered samples where peaks were predominantly at the transcription start site (TSS). Drought and heat-responsive genes and transcription factors were identified within the flanking region of differential peaks. As a comparison between drought and control, the upregulated differential peaks were located predominantly in the promoter and distal intergenic regions. A genome-wide chromatin profiling strategy has been implemented in response to drought in beans to identify nucleosome depleted regions. The understanding of these open chromatin regions will help in identifying potential genomic regions for crop improvement. (The manuscript is being processed for publication) Transcriptomics and Integrated Metabolomics and Proteomics -The data are being processed.
Publications
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Progress 12/15/19 to 12/14/20
Outputs
Target Audience:Graduate and Undergraduate students were trained to conduct field studies. Which includes an experimental plan for the Drought andNon Drought experiments of common bean in a Randomized Complete Block Design. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Students at different levels (undergraduate and graduate students whether funded or not funded) were trained in molecular techniques (DNA and RNA isolation from common bean, cDNA synthesis, polymerase chain reactions, nuclei isolation) and designing the field plan for conducting the drought experiment with six different genotypes, which includes, physiological parameters and using randomized block design for the experimental setup. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Analyzing the sequenced data from the drought experiment conducted under a controlled environment, preparation of RNA, metabolites, methylome, chromatin immune-precipitation of the Nebraska samples. Initiating reference and drought stress transcriptome, epigenome, and metabolomes in the genotypes of common bean grown in Nebraska. Initiating the field study at Delaware in the next summer of 2021 to carry out the drought stress. Preparing the manuscript from the data of controlled drought stress experiment.
Impacts
What was accomplished under these goals?
Field Study Six genotypes of common bean (Phaseolus vulgaris. L) namely(USPT-ANT)x('Matterhornx98078-5-1-5-1),Merlotx(05F-5055-1x98020-3-1-6-2)?,Matterhorn? - Tolerant andMerlot,Stampede, ?Sawtooth? -Sensitive were chosen and grown in two different locations, Delaware and Nebraska. The experiment was conducted in a randomized block design for drought and non-drought treatments. All the genotypes were subjected to drought and non-drought conditions.Uniform soil preparation was done, and the soil was plowed to facilitate the growth of healthy beans.A total of approximately5500 ft²of land wasprepared for this field trial. The land was divided into two sections for Drought andNon-Drought treatments.After field preparation,four rows of 17 ft. lengthwere planted of each genotype in each block. The different entries had been randomized using a random number generator to prevent any bias. The distance between each row was2 ft. The plant to plant spacing was3 inches.Therefore, there were approximately10-15 seeds for every 3.5 ft.Six rows of beans had been planted as a growing border between the Drought and Non-Drought, which did not receiveany irrigation. The distancebetween each entry was4ft. The field trial was similar between the two locations. Tensiometers were also installed in the field to monitor the availability of water in the field to ensure that optimum conditions were maintained at all times. Each genotype was planted in three replications. The experiment was started on the 1st of July 2020. The drought stress was induced after flowering as a terminal drought treatment. Results Delaware The field trial in Delaware was not successful as there was more precipitation during the flowering stage. The precipitation was estimated to be 0.443 inches. The tensiometer reading was shown to be 12 Centibars as the soil was wet. The field trial was not continued as planned in Delaware as there was more precipitation, which affected enforcing the drought stress at the flowering stage. Nebraska The drought treatment was successful at the Nebraska location. The genotypes were subjected to drought stress along with the control non-drought treatment. Young leaves and roots were collected at the end of drought stress. The samples will be processed further to identify the potential genes and their regulation in response to drought stress. Controlled Environment Common beans grown in a controlled environment were subjected to drought stress. The study was carried out to reveal the drought-responsive genomic region of the common bean. Physiological parameters were measured, and the samples are being sequenced in order to reveal the regulatory activity of the genomic region in response to drought stress. Challenges Due to the Covid-19 restrictions and mitigation strategies, there were delays in the experimental process. Limited access to the laboratory further slowed down the pace of experiments.
Publications
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