Progress 10/10/19 to 09/30/20
Outputs
Target Audience:Teaching and laboratory instruction: • One graduate student (PhD) has been recruited to develop her PhD thesis using samples, data and resources from this proposal. Miss Jia Tan enrolled the Integrated Plant and Soil Sciences graduate program at the University of Kentucky, July 2020. Her PhD objectives align with objectives 1, 2, and 4 of this proposal: 1. Determine if epigenetic memory of stress confers grapevine a measurable advantage against future episodes of the same or other stresses. 2. Elucidate the temporal scale of mid-term maintenance of stress-induced epigenetic modifications in grapevine. 4. Compare how different vegetative propagation methods affect the maintenance of epigenetic memory of stress. • PI Lopez has been invited lecturer to two different courses of the University of Kentucky Undergraduate Program Agricultural and Medical Biotechnology (i.e. ABT 101 "Introduction to Biotechnology", and ABT 201 "Scientific Method In Biotechnology") where he discussed the potential applications of epigenetic memory for crop improvement. Extension and outreach: • Invited speaker at the 2020 Kentucky Fruit and Vegetable Conference/Grape and Wine short Course.Title:Epigenetic Memory of Grapevines. Meeting attendants were mainly grapevine growers, winemakers and extension specialists. • Participation at the Round Table Discussion: Grape Pricing, Grower and Winery Connections, and Helpful Tools for The Industry during the 2020 Kentucky Fruit and Vegetable Conference. During this round table, the potential applications of epigenetic priming in grapevines were briefly discussed with participant growers and winemakers. • Invited speaker at the 2020 Annual Meeting of the Argentinian Association of no-till growers (Asociación Argentina de Productores en Siembra Directa - Aapresid) (https://www.aapresid.org.ar/). Title:Epigenetics and Plant Memory • Presentation at the 2020 Kentucky Virtual State Fair.Title:Experimental Vineyard (https://www.youtube.com/watch?v=bu52A4BtCOI&list=RDCMUCjLmlBb3hAPB927mQmijXxw&index=1). Changes/Problems:Covid19 pandemic has slowed down the laboratory work (DNA methylation analysis could not be performed) and analysis of the generated results (Gene expression analysis could not be completed). However, we believe that these delays should not affect significantly the expected outcomes of the project. What opportunities for training and professional development has the project provided?Miss Jia Tan through this project has acquired skills in molecular biology and bioinformatics. Miss Tan has presented the results of her work during Dr Lopez weekly research group meetings and did a complete presentation of her progress at her annual PhD committee meeting which has helped her building her communication skills. Additionally, this project is closely related toUSDA-NIFA project "Implications of epigenetic rejuvenation during vegetative propagation for the production of locally adapted perennial crop cultivars" accession Number: 1018617.This project involves the participation of two graduate students and one postdoctoral researcher. It is expected that the results from both will finally be integrated to generate a cohesive piece of research. Collaboration with other lab members for the purpose of this project also has help to enhance her communication skills. In addition to the Integrated Plant and Soil SciencesGraduate Program 2019-2020 courses, MissTan attended the METHADA 2020 eHands on - Virtual Training School in Transcriptomic Metadata Handling and Data Analysis (http://www.integrape.eu/index.php/training-schools/methada-virtual)as a registered observer. This training school addresses grapevine genomics data handling and analysis, and it is organized in three modules. 1. On the first unit, trainees will work to learn how to use the guidelines provided by the INTEGRAPE community to correctly annotate experiments and submit them on public repository. We will explore standards and bio-ontologies for FAIR data annotation using their personal data. 2. The second module will teach the trainees how to use the tools from Sequentia (AIR for transcriptomics). 3. Finally, attendees will be introduced to the GREAT (GRapevine Expression ATlas) platform for transcriptomic meta-analysis. The GREAT platform is a good example of reusing publicly available RNA-seq data to answer new biological questions. Trainees will have the opportunity to explore the GREAT platform with their own list of genes of interest Graduate Student associated to this project Miss Jia Tan, has applied for two competitive grants (SouthernSAREGraduate StudentGrantprogram and University of Kentucky College of Agriculture Food and the Environment Graduate Student Research Activity Award). Beyond the obvious benefits of successfully applying for a competitive grant as Graduate Student, both applications help Jia develop grantmanship skills that will be of great use as her career as plant scientist develops. How have the results been disseminated to communities of interest?• Invited speaker at the 2020 Kentucky Fruit and Vegetable Conference/Grape and Wine short Course.Title:Epigenetic Memory of Grapevines. Meeting attendants were mainly grapevine growers, winemakers and extension specialists. • Participation at the Round Table Discussion: Grape Pricing, Grower and Winery Connections, and Helpful Tools for The Industry during the 2020 Kentucky Fruit and Vegetable Conference. During this round table, the potential applications of epigenetic priming in grapevines were briefly discussed with participant growers and winemakers. • Invited speaker at the 2020 Annual Meeting of the Argentinian Association of no-till growers (Asociación Argentina de Productores en Siembra Directa - Aapresid) (https://www.aapresid.org.ar/).Title:Epigenetics and Plant Memory • Presentation at the 2020 Kentucky Virtual State Fair.Title:Experimental Vineyard (https://www.youtube.com/watch?v=bu52A4BtCOI&list=RDCMUCjLmlBb3hAPB927mQmijXxw&index=1). What do you plan to do during the next reporting period to accomplish the goals?1. DNA methylation analysis of samples collected during year 1 will be carried out using Reduced Representation BIsulfite Sequencing and Whole Genome BIsulfite Sequencing.. 2. Gene expression analysisof samples collected during year 1 will be finalized. 3. Phenotypes, methylomes and transcriptomes of control and stressed plants (obtained in Phase I) will be comparedto investigate themaintenance of memory of stress (Objective 2). In parallel, the flux of stress induced DNA methylation changes will be assessed at a genome level to identify the regions/context or genomic features that maintain/lose over time(Objective 3). 4.To determine if exposure to stress confers grapevine a measurable advantage against future episodes of stress (objective 1) and the effect of vegetative propagation on epigenetic memory (Objective 4), naïve mother plants and propagules (grown under control conditions during year 1,and primed mother plants and propagules(grown under stress conditions during year 1) plants will be divided into four groups so that half of each cohort is exposed to both conditions described for year 1 (e.g. Naïve plants grown under control conditions on year 2 and naïve plants grown under stress conditions on year 2. Same for primed plants). Plants will be characterized both phenotypically and molecularly as described above.
Impacts
What was accomplished under these goals?
95 Vitis vinifera (L.) Carbernet Sauvignon plants were grown under T0: control conditions T1: water stress T2: high-temperature stress T3: dual stress Samples were collected at 6time points ST1: 1st day plant acclimation to glasshouse ST2: 1st day of drought treatment ST3: 11th day of drought treatment and 1st day of heat treatment ST4: 12th day of drought treatment and 2nd day of heat treatment ST5: Day of stress removal ST6: Plant physiological recovery, 16 days after removal of stress treatments All plants were phenotypically characterized prior, during and after treatment by measuring stomatal conductance, stem water potential, and leaf temperature. Stomatal conductance (Gs) differences between control and stressed (drought, heat and drought/heat) plants varied depending on the stress and the sampling times measured. At ST2, no significant differences were observed between control and drought plants. At ST3 (11 days into drought stress and first day of heat treatment), heated plants showed significantly higher Gs than control plants (t-test, P<0.01). On the contrary, plants under drought and drought/heat conditions significantly reduced their Gs by approximately 90% (t-test, P<0.01). At ST4 (12 days into drought stress and second day of heat treatment) heated plants showed no significant differences in Gs when compared to control plants, while plants under drought and drought/heat conditions maintained the observed reduction in stomatal conductance at ST3 (t-test, P<0.05). No significant differences in Gs were observed at any time point between drought and drought/heat plants. After the removal of stress at ST5 the Gs of plants under heat stress showed a significantly higher level of stomatal conductance than control plants (t-test, P<0.05), while those under drought and drought/heat stress did not show significant differences. Stressed plants recovered gradually until achieving complete recovery at ST 6. Plants under drought/heat stress showed a relatively faster recovery than plants under drought stress. Stem water potential (stemΨ) shows a negative effect of drought and heat stress on the grapevine water status. In brief, the stemΨ of control plants stayed at around -0.4 MPa during all the experiment stage. At ST 2 (i.e. first day of drought treatment), a significant decline in stemΨ was observed for drought-treated plants (t-test P<0.05). During drought and heat treatment (ST3, ST4), the stemΨ of the single heat-treated plants experienced an significant decrease (t-test, P<0.001), from -0.4 MPa to -0.75 MPa. As the drought treatment progressing, the stemΨ of the single drought-treated plants continue to drop from -0.57 MPa to -0.76 MPa, showing an extremely significant difference from control (t-test, P<0.001). Plants under drought/heat treatment showed the lowest stemΨ at approximately -0.97 MPa (t-test, P<0.001). After stress removal at ST 5, the stemΨ of heat-treated plants showed no significant difference. However, the stemΨ of drought-treated plants and drought/heat-treated plants was still significantly different from that of control at ST5 (t-test, P<0.001). At ST6 (16 days after removal of both stresses), no significant differences in stemΨ were observed between the treated plants and control. No significant difference was observed in leaf temperature between drought-treated plants and control over all six sampling times. However, some different situations were observed between the heat-treated and drought/heat-treated plants. At ST2, no significant difference was displayed between control and treatments. At ST3, the leaf temperatures of both third young leaf and first fully expanded leaf showed significant differences between control and drought/heat treatment (t-test, P<0.001). However, heat-treated plants showed significant difference only in the third young leaf (t-test, P<0.001). At ST4, the leaf temperatures of both treatments continued to go up. The third young leaf temperature of heat-treated and drought/heat-treated plants peaked at 38.1 °C and 40 °C, respectively. In addition, the first fully expanded leaf temperature of heat-treated and drought/heat-treated plants peaked at 36.1 °C and 42 °C, respectively. At ST5, the third young leaf temperature of heat-treated plants decreased significantly and no significant differences were observed between other treatments and control. At ST6, the leaf temperature of all plants was around 30 °C, showing no significant difference among treatments. Leaf tissue was collected from all plants to compare gene expression (whole transcriptome sequencing) and DNA methylation (reduced representation bisulfite sequencing (RRBS)) profiles of stress and control plants prior, during and after treatment. DNA and RNA extractions were performed for all samples. Next Generation Sequencing results was performed to characterize differences in gene expression between treatments and time points. screened for the identification of differentially methylated and/or expressed genes using in house and freely available software. Multidimensional scaling (MSD) of the total transcriptome results was used to visualize the differences in transcriptional profiles among all 84 samples. MDS plot shows that the gene expression grouped samples by treatments and by sampling times. Briefly, control plants, before treatment, during treatment and after treatment occupied the top quadrants of the MDS plot. Samples collected from stressed plants during ST3 and ST4 occupied the furthest distance from samples collected during ST1 and ST6. Finally, samples collected from stressed plants under the first day of each stress (ST2 for drought stress and ST3 for heat stress) and the first day of the stress condition removal (ST5) occupied an intermediate space between samples at ST1/ST6 and ST3/ST4. To interpret and analyse the RNA-seq data, the differential expression of transcripts were examined between treatments and control (drought vs. control, heat vs. control, drought/heat vs. control). The differentially regulated geneswere identified for the drought, heat and drought/heat treatments (Table 2). Differential expression analysis revealed that the majority of up-regulated and down-regulated genes were identified in the drought/heat treatment. Under drought stress, the number of DEGs showed an increase at ST2 and ST3 followed by a decrease at ST4. No DEGs were detected at the day of stress removal (ST5). The number of up-regulated genes increased from 23 at ST2 to 110 at ST3 and then dropped to 36 at ST4. Conversely, the number of down-regulated genes decreased from 37 at ST2 to 11 at ST3 and then raised to 30 at ST4. In terms of heat treatment, the majority of DEGswere found atST4. The number of up-regulated DEGs varied from 3 at ST3 to 81 at ST4 whereas the number of down-regulated DEGs varied from 23 at ST3 to 69 at ST4. At ST5 (Day of removal of stress treatments), only one up-regulated gene and one down-regulated gene were obtained respectively. Under drought/heat stress, the up-regulated and down-regulated genes of drought/heat treatment both showed a dramatic increase in number. The number of up-regulated DEGs increased from 236 at ST3 to 882 at ST4 while the number of down-regulated DEGs went up from 83 at ST3 to 1236 at ST4. At ST5, 75 up-regulated genes and 71 down- regulated genes were expressed differentially. At ST6, there were still two up-regulated DEGs in drought/heat treated plants. Further analysis is currently been carried out to determine the gene networks and functions associated to stress response in grapevine. All plants were propagated using either dormant callus or layering. Propagules and mother plants were kept in order to study the maintenance of epigenetic memory of stress after vegetative propagation during subsequent years.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2020
Citation:
Kendall R. Corbin and Carlos M. Rodriguez Lopez (January 29th 2020). Library Preparation for Whole Genome Bisulfite Sequencing of Plant Genomes, DNA Methylation Mechanism, Metin Budak and Mustafa Yildiz, IntechOpen, DOI: 10.5772/intechopen.90716. Available from: https://www.intechopen.com/books/dna-methylation-mechanism/library-preparation-for-whole-genome-bisulfite-sequencing-of-plant-genomes
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