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INVESTIGATOR: Zhou, S.

PERFORMING INSTITUTION:
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE, TENNESSEE 37209

TRANSGENERATIONAL ADAPTATION OF PLANTS TO ACIDIC PH AND TOXIC METALS IN SOIL

NON-TECHNICAL SUMMARY: This collaborative research project (Tennessee State University, Delaware State University and USDA/ARS) is aligned with Program Priority Area g: Sustainable Agriculture and addresses CBG need areas: Advancing Agricultural Sciences, and secondly: Building a Centralized Research Support Systems. Our earlier studies on aluminum (Al)-treated tomato (Solanum lycopersicum) plants have shown that progenies from successive generations grown under sub-lethal Al challenged conditions produced successively larger and more developed root systems, compared to those from non-treated parental plants. To understand this phenomenon we propose to develop the research capacities to investigate and utilize the transgenerational plasticity observed and to build a research leadership team to utilize the science and technology discovered to advance sustainable agriculture. To these ends, we propose to: 1) identify Al and acidic stress-induced methylomes, transcriptomes and proteomes in tomato plants; 2) develop an understanding of the molecular mechanisms which drive the process toward transgenerational adaption to these stress factors, through an integrated analysis of these omics data; and 3) implement a Tomato Cyber Lab for managing and sharing of the omics data in compliance with Findable, Accessible, Interoperable, and Re-usable (FAIR) principles, and for participants to have secure access to instruments in real-time allowing hands on learning from geographically distant locations. The outcomes of the proposed research will include new scientific knowledge for improving plant resilience against environmental stress, enhanced skill sets, expertise and research prowess among the faculty and students of two premier 1890 Universities, with anticipated impacts on an increased capacity to develop sustainable agricultural programs.

OBJECTIVES: This is a collaborative project among Tennessee State University (TSU), Delaware State University (DSU), and USDA-ARS (Cornell). The goals of this proposal are: 1) to build research capacities for investigating molecular mechanisms underlying transgenerational adaptation to aluminum (Al) and acid stress observed in tomato plants, 2) to develop a Cyber-based platform for increasing the efficiency in conducting research and broadening the pool of researchers who will deliver the science and technology derived from the project to promote sustainable agriculture.The project has three objectives:To identify Al and acid stress-induced methylomes (DSU), transcriptomes (TSU) and proteomes (USDA) in tomato plants showing transgenerational adaptation phenotypes; To develop an understanding of the molecular mechanisms which drive the transgenerational adaption process, through an integrated analysis of these omics data;To implement a Tomato Cyber Lab for managing and sharing of the omics data in compliance with Findable, Accessible, Interoperable, and Re-usable (FAIR) principles , and for participants to have access to resources at geographically distant locations.

APPROACH: Tomato 'Microtom' plants derived from seeds pre-exposed to Al/acidic pH conditions from 0-4 generations will be subjected to the same treatment conditions as their parental plants. The control solutions will be prepared at pH 6.0. These treatments will be applied for 30 days. Physiological data including plant photosynthesis will be collected during the treatment period. At the end of the treatment experiment, plant size, root size, mineral contents, and Al accumulation on root-tips will be recorded. These data will be used to analyze the physiological properties asssociated with transgenerational effects.The distal 1cm root tips from primary and lateral roots will be harvested and these tissues will be used for all the analysis to be carried out in this project. Three biological replicates will be included for each treatment conditionper seed types.Three analyses will be conducted: methylome and transcriptome profiling and quantitative proteomics. Methylome analysis will use the whole-genome bisulfite sequencing (WGBS). The dfferentially methylated cytosines (DMC) between sample pairs, the CG, CHG, and CHH contexts will be analyzed using the Fisher's exact test. DMCs with a distance of <100 bp apart and at least 4 DMCs will be merged into differentially methylated regions (DMRs), when methylation levels differed by 0.4, 0.2, and 0.1 for CG, CHG, and CHH, respectively. All the CpG islands, gene promoters, and most genetic regulatory elements, gene bodies, and repeated DNA sequences will be identified. Transcriptome analysis will be conducted using the next generation RNA sequencing technology.TMT Proteomics analysis will be conducted to identify expression of proteins in each sample.Differentially expressed genes (DEGs), Differential expressed proteins (DEPs) and DMRs in same pairwise comparisons will be identified. The association between DMGs (genesoverlappingDMRs) and DEGs, DEPs will be estimated by Pearson correlation test. The following five modules will be developed: 1). Induced-DMRs and Repressed DEGs/ DEPs, for a negative correlation; 2). Induced-DMRs and Induced DEGs/ DEPs, for a positive correlation; 3). Repressed-DMRs and Induced DEGs/. DEPs, for a positive correlation; 4). Repressed-DMRs and Repressed DEGs/ DEPs, for a negative correlation; 5). No significant correlation between these different types of gene regulation modulation. The weighted gene co-expression network analysis (WGCNA) algorithm will be used to identify gene behavior modules associated with transgenerational adaptation to Al and acid, or a combination of both stress factors. Gene members in the modules identified above will be searched in gene ontology (GO) terms. Gene lists under the same GO terms derived from protein/transcriptome modules and transcriptome/methylome modules will be merged. Finally causal models from methylation to transcripts to proteins, and those associated with enriched functions in stress tolerance will be developed. Ultimately, we will be able to deliver the epigenome and the gene and/or protein co-expression models that are responsible for establishing transgenerational adaptation to acid and Al stresses. A web-based secure data sharing and data management system will be implemented. A cyber lab for data sharing and analysis will be established. Student research leadership will be built by training graduate and undergraduate students with skills and aptitude in self-motivation, creativity, and team collaboration. The USDA cooperator and 1890 senior faculty will mentor young faculty in developing research projects and student advisement.

PROGRESS: 2019/04 TO 2020/03
Target Audience:The target audiences reached through this project include the following groups: Graduate and undergraduate students. The participating faculty and post-doc on this project have mentored graduate and undergraduate students. These students conducted research projects on Al stress. Faculty has used the research materials to teach 'Plant Propagation' classes. Science community. This project has produced four journal publications in this reporting period. These papers have been read and cited. This project has been also introduced to the scientific communities through presentations and information exchange from project participants and peers. Changes/Problems:The tornado outbreak of March 2-3, 2020 in middle Tennessee made a significant damage to all the buildings on the Agricultural Research Station, Tennessee State University. At that time, we were conducting tomato treatment experiment using the hydroponic system in a greenhouse. The tornado torn off the roofof the greenhouse and desrupted the heating and power supplies, and the experimental plants were injured from cold temeperature. Then theCOVID-19 pandemic slowed down everything.Theseincidences have delayed many project activities. Still, the project team is making all the efforts possibleto try to accomplish the project goals in a timely manner. What opportunities for training and professional development has the project provided?At Tennessee State University, the project supported two senior faculty to attend 2019 ASHS Annual Conference and ASPB Plant Biology 2019, when they madepresentations of the project results. Faculty, post-doc and undergraduate students attended the 19th Biennial Research Symposium Association of 1890 Research Directors, 3.30-4.3.2019, Jacksonville, FL.TSU faculty (2), graduate (1) and undergraduate students (2), post-doc research associate (1) attended two training sessions for proteomics at the USDA-ARS labs. The project provided training in tomato hydroponics culture, plant physiology, tissue harvest and lab analysis to 15 graduate students, six undergraduate students, and three cohorts (50) of high school students from Metro Nashville Public Schools and three high school Ag science teachers. A senior research scientist at USDA-ARS attended a professional conference. How have the results been disseminated to communities of interest?We have used publications on scientific journals, presentations at professional conferences, lectures in graduate and undergraduate classes, outreaches to local communities to disseminate the project results. What do you plan to do during the next reporting period to accomplish the goals?At the present time, we have collected all the tissue samples, and started sequencing transcriptomes and proteomics analysis.In the next reporting period, we plan to complete analysis of methylomes, transcriptomes and proteomes; and develop the cyber labs for data sharing and access for all the participating labs.

IMPACT: 2019/04 TO 2020/03
What was accomplished under these goals? 1. Plant treatment and tissue collection. First, we have generated eleven seed types for tomato 'Micro-Tom'. These seedsinclude G0; Al-G1,G2,G3,G4,G5; Al+G1,G2,G3,G4,G5. G0 seeds were harvested from tomato plants grown in Magnavaca's hydroponic culture, pH 5.8; Al- G1-G5 seeds were derived from plants grown in acidic (pH4.5-4.8) Magnavaca's hydroponic solutions for 1-5 generations; and Al+ G1-G5 seeds were derived from plants grown in acidic Magnavaca's solution supplemented with Al (100 µM AlK(SO4)2•12H2O) for 1-5 generations. For plant treaments, these seeds were seeded in rock wool seed cubes inserted in netted pots which were placed in Magnavaca's hydroponic solutions. Three treatments were set-up as: pH 5.8; Al-, pH 4.5; Al+ , pH 4.5. Only G0 plants were grown in the pH 5.8 treatment solution. Three to four hydroponic tanks (each serves as one replicate) were included for each seed type per treatment condition. Root-tips (the basal 1 cm fragments) were collected for short-term treatments (prior to flowering stage) and long term treatments (plants bearing tomato fruits). The plant root size/ root length were measured. Leaves, roots and stems were harvested, dried, and stored for mineral analysis. 2. For tissue analysis, proteomics analysis for long-term treated root-tip tissues (total of 90 samples) have been completed. This is the first time we used the TMTpro 16plex Label kits (ThermoFisher Scientific, MA). For transcriptome analysis, the 90 long-term treated samples have been processed including RNA extraction and library preparation using TruSeq Stranded mRNA kit with IDT for Illumina-TruSeq RNA UD indexes 96 indexes (Illumina, CA), sequencing using Illumina NextSeq, and data analysis. Genomic DNA of these samples were sent to Delaware State University for methylome analysis.

PUBLICATIONS (not previously reported): 2019/04 TO 2020/03
1. Type: Journal Articles Status: Published Year Published: 2020 Citation: Shaolan Yang, Hui Li, Sarabjit Bhatti, Suping Zhou, Yong Yang, Tara Fish, Theodore W. Thannhauser, 2020. The Al-induced proteomes of epidermal and outer cortical cells in root apex of cherry tomato âLA 2710â, Journal of Proteomics, 211:103560.
2. Type: Journal Articles Status: Published Year Published: 2020 Citation: Xiudong Sun, Hui Li, Santosh Thapa, Sasikiran Sangireddy, Shaolan Yang, Xiaobo Pei, Wei Liu, Yuping Jiang, Sarabjit Bhatti, Yong Yang, Tara Fish, Theodore Thannhauser, shaolan yang, and Dafeng Hui. 2020. Al-induced proteomics changes in tomato plants over-expressing a glyoxalase I gene. Hortic Res 7, 43. http://sci-hub.tw/10.1038/s41438-020-0264-x
3. Type: Journal Articles Status: Published Year Published: 2020 Citation: Hui Li, Joshua OâHair, Santosh Thapa, Sarabjit Bhatti, Suping Zhou, Yong Yang, Tara Fish, Theodore W. Thannhauser. 2020. Proteome profile changes during poly-hydroxybutyrate intracellular mobilization in gram positive Bacillus cereus tsu1. BMC Microbiol 20, 122. http://sci-hub.tw/10.1186/s12866-020-01815-6.
4. Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhujia Ye, Sasikiran Sangireddy, Chih-Li Yu, Dafeng Hui, Kevin Howe, Tara Fish, Theodore W. Thannhauser, and Suping Zhou. 2020. Comparative Proteomics of Root Apex and Root Elongation Zones Provides Insights into Molecular Mechanisms for Drought Stress and Recovery Adjustment in Switchgrass. Proteomes. 8, 3; doi:10.3390/proteomes8010003.
5. Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Suping zhou. Identification of proteomes in Al-sensitive root tip cells of cherry tomato âLA 2710â. Plant Biology 2019
6. Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Candice C. Haymon-Gresham, Jesse Potts, Hui Li, Suping Zhou,. Effects of multiple-generation exposure to Al treatments on tomato plant responses to toxic ions. 42nd Annual Tennessee State University-Wide Research Symposium
7. Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Jesse Potts Candice C. Haymon-Gresham, Hui Li, Suping Zhou.Effect of recurrence of Al toxicity stress on tomato plant growth. 42nd Annual Tennessee State University-Wide Research Symposium