Progress 09/01/10 to 08/31/14

Outputs
Target Audience: this project has resulted in the graduation of one master and one Ph.D. students, povided training to over ten graduate and undergraduated students and produced five journal publications, two dissertations and one theses, 18 conference proceedings and several brochures. Six awards were receivedfor our publications from student conference presentations and for travel funding. This grant has supported the development of research capacity and functional genome studies of abiotic stress in tomato and other plant species which includes greehouse treatment , gene cloning and gene knock-down procedures. Activities performed through this project has played a critical role in steering the research endeavor toward adopting cutting-edge technologyies and producing quality students in the field of agriculture. The impacts of this project: Aluminum toxicity is a major problem affecting crop productivity in acid soils. The 4 years research activities during the grant period have provided large amount of information on the molecular mechanisms affecting plant tolerance to Al, in particular in protein expression. Findings from this research have been disseminated to a large number of audiences. Two publications in Hort-science testify to the improtance of our studies on the toxic effects of Al in tomatoes. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Funding of this project has funded the research projects of one master and two Ph.D. students. It as also provided for the supplies and equipment for the training of more than ten graduate and undergraduate students. Five journal publication, two dissertations and one master's thesis and 18 proceedings were published. Six awards were earned through journal articles, student presentations and travel grants for graduate students to attend and present their research results at scientific meetings.. How have the results been disseminated to communities of interest? Yes, just about everything discovered were published. Dr. Suping Zhou was invited to present lectures at Nanjing Agricultural University, that the Shanghai Vegetable Research Institute, and at the Beijing Agricultural University to present the work on abiotic stress in tomato caused by Alulminum toxicity What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? All objectives listed above were accomplished. Mineral analysis fond that Al2(SO4)2 treated tomato contained a significant higher amount of Al in roots (29-fold), and in green and orange and red tomato fruits (2-fold). Morin (2', 3, 4,5, 7-pentahydroxyflavone) staining also detected a higher flueorescence intensity in Al treated roots. In the relative and absolute quantitation (iTRAQ) proteomic analysis, 1442 proteins were identified in roots, of which 34 proteins showed significant changes in abundance between the Al-treated and control tidsdsues. In green, orange and red tomato fruits, 124 of 1133 proteins, of which 34 proteins showed significant changes in abundance between the Al-treated and control tissues. in green, orange and red tomato fruits, 124 of 1133 proteins, 39 of 1362 proteins, 44 of 1362 proteins showed significant changes in abundance between Al-treated and control tissues in each tissue type, respectively. Trnagenic tomato 'Money Maker' over expressing a lactoylglutathione liase gene and FHA1 homolog were generated. Three transgenic lines were confirmed to contain the insert gene. These trangenic lines are currently being evaluated for tolerance to Al toxicity. The two publications in the J. Amer. Hort Society received the paper of the year award. The J. Amer. Soc. Hort. Sci. 138(5):382-394 was sited and the most outstanding paper on horticultural cross-commodity research, including broadly applicable methodology, pre- and postharvest physiology, genetics, anatomy, morphology, ecology, crop production systems, modeling and management published by the Society in the year 2013. The paper published in J. Amer. Soc. Hort. Sci. 136(4):288-302 received the ASHS Vegetable publication award in recognition of the most outstanding paper on vegetable crops published by the society in the year 2011.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhou, Palmer, Zhou, Bhatti, Howe, Fish, Thannhauser. Differential root proteome expression in tomato genotype with contrasting drought tolerance exposed to dehydration. Journal of the American Society for Horticultural Science. 138:131-141.
• Type: Theses/Dissertations Status: Accepted Year Published: 2013 Citation: Sangireddy, S. R. PhD's dissertation. Identification of molecular and physiological changes in tomato in responses to aluminum stress and functional studies of aluminum responsive genes. Tennessee state University, 2013.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Zhou, Sauve, Liu, Sangireddy, Bhatti, Hucko, Fish, Thannhauser. Identification of salt-induced changes in leaf and root proteomes of the wild tomato, Solanum chilense. J. Amer. Soc. Hort. Sci. 136:288-302.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Nveawiah-Yoho, Zhou, Palmer, Sauve Zhou, Howe, Fish, Thannhauser. Identification of proteins for salt tolerance using a comparative proteomic analysis of tomato accessions with contrasting salt Tolerance. J. Amer. Soc. Hort. Scie. 138:382-394.
• Type: Theses/Dissertations Status: Accepted Year Published: 2013 Citation: Okekeogbu. Aluminum accumulation and its effects on the acquired tolerance and proteome expression in tomato seeds during seed maturation. MS thesis, Tennessee State University. ProQuest/UMI dissertation publishing (UMI #10531).
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Sauve & Zhou. Aluminum induced phyusiological and molecular regulations in 'Micro Tom' tomato roots. The 10th Solanaceae Conference. Oct 13-17, 2013 Beijing, China
• Type: Theses/Dissertations Status: Accepted Year Published: 2013 Citation: Nveawiah-Yoho. Identification of proteins for salt tolerance using a comparative proteomic analysis of tomato accessions with contrasting salt Tolerance.Ph.D. Dissertation. Tennessee State University.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Zhou. Comparative proteomics analysis of tomato varieties with contrasting tolerance traits reveals root proteins for salt and or water deficit tolerance. The 210T Solanaceae Conference. Oct. 13-17. Beijing, China.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Okekeogbu, Sangireddy, Li, Bhatti, Zhou, Howe, Fish, Yang, Thannhauser. Effect of aluminum treatments on proteomes in radicles of seeds derived from Al-treated tomato plants. Proteomes 2:169-190.

Progress 09/01/12 to 08/31/13

Outputs
Target Audience: During this grant period, this project has produced one journal paper and five abstracts. The students and scientists working on this project have made seven presentations at TSU in-house seminars, regional, national and international meetings. Through this effort, the project has reached audiences including stake holders in horticultural industry, graduate and undergraduate students and scientists in both basic and applied research area. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? For student training, three TSU Ph.D. and Master students attended 2013 summer internship at the USDA/ARS laboratory on Cornell campus and the proteomics workshop and confocal microscopy training at Cornell University. The project supported students to participate in presentation competition at national conferences. One student have completed research thesis on this project and have received master degree from Tennessee State University. One Ph.D student has completed research and is in the process preparing thesis to graduate from TSU. How have the results been disseminated to communities of interest? During this grant period, this project has produced one journal paper and five abstracts. The students and scientists working on this project have made seven presentations at TSU in-house seminars, regional, national and international meetings. Thesis of Master degree for one graduate student was published. What do you plan to do during the next reporting period to accomplish the goals? 1. The Ph.D student will complete preparation of dissertation, and publish the research paper in referred journals; 2. Repeat aluminum treatment experiment, and publish the results together with a proteomics analysis performed in Summer, 2013; 3. Test transgenic tomato plants for tolerance to aluminum and chromium and selecting functional genes.

Impacts
What was accomplished under these goals? During this project period, we focused on the analysis of Al-accumulation in different parts of tomato plants, and the corresponding proteome expression. Tomato plants were subjected to Al- treatment for different durations. Al content in roots, leaves and fruits at different maturity stages were analyzed using ICP and ICP-MS, and morin staining methods. Total proteomes of collected tissues were analyzed using iTRAQ and DIGE-MS methods. Transformation vectors were constructed and transgenic tomato plants over-expressing several Al-related genes have been regenerated. The ongoing experiments include screening for homozygous transgenic plants and characterization of their tolerant to Al and chromium ions. Experimental results were disseminated via presentations made by participating scientists and students at departmental seminars and regional, national and international conferences. Several papers have been published from the projects. For student training, three TSU Ph.D. and Master students attended 2013 summer internship at the USDA/ARS laboratory on Cornell campus and the proteomics workshop and confocal microscopy training at Cornell University. The project supported students to participate in presentation competition at national conferences.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Suping Zhou,Marsha Palmer, Jing Zhou and Sarabjit Bhatti, Kevin J. Howe, Tara Fish and Theodore W. Thannhauser. Identification of Proteins for Salt Tolerance Using a Comparative Proteomics Analysis of Tomato Accessions with Contrasting Salt Tolerance. JASHS, 138:131-141. Ikkenna Okekeogbu, Sasikiran Reddy Sangireddy, and Suping Zhou. 2013. Proteome Expression of Maturing Tomato Seeds under Aluminum Stress. Post-transcriptional Gene Regulation in Plants meeting. July 25-26. Ikkenna Okekeogbu, Sasikiran Reddy Sangireddy, and Suping Zhou. 2013. Evaluation of Aluminum Induced Tolerance and Proteome Changes in Tomato Seeds. Plant Biology Annual Meetings, July 20-24. Sasikiran Reddy Sangireddy, Sarabjit. Bhatii, Ikenna Okekeogbu, Suping Zhou, Roger Sauve, Theodore W. Thannhauser. 2013. Characterization of Lactoylglutathione lyase and FHA1 homolog genes for aluminum stress. Providence, Rhode island. July 25-26. Sasikiran Reddy Sangireddy and Suping Zhou. 2013. Proteomic analysis of micro tomato roots under aluminum stress (Oral Presentation). 17th Biennial Research Symposium (ARD) conference. Jacksonville, FL. 2013 March 9-15. Sasikiran Reddy Sangireddy and Suping Zhou. 2013. Aluminum stress in Micro Tomato Roots: A Study of the Physiological and Molecular Regulations. 33rd Annual University-Wide Research Symposium. TSU, Nashville, TN. 2013. March 2-7.

Progress 09/01/11 to 08/31/12

Outputs
OUTPUTS: During this project period, we have been focusing on set-up an aluminum treatment system using Micro-Tom tomatoes, determination of aluminum distribution in different parts of tomato plants, identification of proteins that are affected by aluminum ion toxicity, and making transformation constructs and transgenic tomato plants. We have acquired different transformation vectors from Purdue University and Cornell University, and are in the process of setting up a more efficient system in the laboratory. We have also initiated the research on virus-induced gene silencing system for functional analysis of the identified genes, vectors have been obtained from different laboratories, and tentative collaboration with PI's from other universities have been agreed upon. For project disseminations, two presentations have been made to share the research findings with scientific communities. The PI has also made several in-house presentations on school campus. about the research activities of this project. The students have made significant progresses on their research thesis. PARTICIPANTS: Kevin Howe, Tara Fish, Theodore W. Thannhauser, USDA-ARS; Graduate students at Tennessee State University: Sasikiran Reddy, and Okekeogbu, Ikenna TARGET AUDIENCES: Scientific community, tomato growers,and students PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A hydroponic system for aluminum treatment experiment on tomatoes have been set up. Mineral analysis confirmed that the toxic ion was accumulated in large amount in roots and leaves. The content in the fruit tissues was low and not consistent among the biological replicates. Using another technique (Morin staining), we have observed deposition of aluminum in fruits and seeds. A more sensitive technique (such as ICP-MS) will be used for aluminum quantification in the next experiment. Protocols for protein extraction from root, leaf, green and red fruits have been developed. The iTRAQ proteomics analysis of root proteins has been completed. Meanwhile, a statistical analysis procedure for analyzing iTRAQ data was developed in collaboration with statisticians. This will be used for future analysis of other samples. Permits for handling transgenic materials have been approved by APHIS. Several transgenic constructs have been prepared, and one transgenic line has been developed. Student training: One Ph.D. student and one master-level graduate student have successfully set up the procedure for making gene constructs for tomato transformation. They both have received training on proteomics analysis at the USDA-ARS laboratory in Summer, 2012 while conducting researches on this project.

Publications

• Sangireddy, S. R., Zhou,S., Bhatti, S. and Sauve, S. 2012. Using Micro-Tom Tomato as a Landscape Plant. 57th Annual SNA Horticultural Research Conference,
• Liu,Z., Sauve, R. and Zhou, S. 2012. Identification of Molecular Mechanism for Freeze Tolerance in Flower Buds of Helleborus orientalis. Plant Stress 6 (1), 1-7

Progress 09/01/10 to 08/31/11

Outputs
OUTPUTS: Tomato cDNA clones were obtained from the Kazusa DNA Research Institute, Tokyo, Japan. The sequence of these cDNA clones were confirmed and used to make over expression transformation constructs. To date, one gene was cloned onto the pRCS2 binary vector and transferred into Agrobacterium tumefaciens strain LA4404. This vector is now ready for performing genetic transformation of tomato. One Doctoral student was recruited and trained to work on this project and two master level graduate students were trained on ITRAQ-proteomics at the USDA collaborator's (Dr. Thannhauser) lab during summer 2011. PARTICIPANTS: Graduate students: Sasikiran Reddy, Jing Zhou, Marsha Palmer. Dr. Thannhuser at Cornel University provided proteomics training in his laboratory to two TSU graduate students, Jing Zhou and Marsha Palmer during summer 2011. TARGET AUDIENCES: Basic research has been accomplished that will serve as the foundation for the project. On refereed journal publication has been written and published. PROJECT MODIFICATIONS: No major changes are needed at this time, the progress of this project is on time.

Impacts
The technical system for making genetic transformation of tomato has been developed and gene clones for testing their functions for tolerance to toxic ions have been sequenced. These accomplishments are the foundation for functional studies of selected genes in transgenic tomatoes. The Doctoral student has been trained and is ready to write his disertation research proposal. One graduate student has completed her research and will begin to write the thesis for a Master's degree.

Publications

• Zhou, S., Sauve, R. J., Liu, Z., Reddy, S., Bhatti, S., Hucko, S.D., Fish, T. and Thannhauser, T. W. 2011. Identification of Salt-induced Changes in Leaf and Root Proteomes of the Wild Tomato, Solanum chilense. J. Amer. Soc. Hort. Sci. 2011 136: 288-302.