Progress 09/01/05 to 08/31/10
Outputs
OUTPUTS: During this period, we have identified proteins associated with aluminum and salt toxicity in tomatoes.The research results have been disseminated to the scientific communities through publications and seminary presentations. We have received requests for reprints from scientists in many foreign countries such as India, China,and Belgium . By responding to these inquires, we have shared the gained knowledge from this research with other scientists working in this area of research. Findings from this research project were shared with Solanaceae community through an invited presentation at the 6th Solanaceae Genome Workshop (SOL 2009, New Delhi, India - NOVEMBER 08-13). The PI also used tomato plants and the developed molecular techniques in a graduate course. These plants as well as the molecular techniques were also used to train graduate students. Through this project,two graduates students were trained. Presently, we are in the process of setting up a proteomics research facility at TSU for the purpose of demonstrating and training this technology to scientists and students. It is our expectations that the establishment of a protonomic facility will encourage other faculty members to adopt this modern powerful molecular technology in their teaching and research projects. PARTICIPANTS: Students at TSU: Zong Liu, Ph.D. student Sashi Ready, Ph.D. student; Peter Yoho, Ph.D. student, Jing Zhou, undergraduate student. Collaborator: Theodore William Thannhauser, PLANT, SOIL AND NUTRITION RESEARCH UNIT - RM 102A TOWER RD. ITHACA, NY, 14853-2901Ted.Thannhauser@ars.usda.gov Phone: (607) 255-8808 Fax: (607) 254-6510. The PI and graduate students participated in two proteomics training sessions at the USDA laboratories, and a tomato genome sequence seminar workshop at Cornell University organised by Dr. Lukas Mueller during the summer of 2010. In addition, two graduate students attended the 2010 Plant and Animal Genome Conference in San Diego, California. TARGET AUDIENCES: The training of graduate students on the use and appllication of novel molecular and bioinformatics tools in agricultural research was the main target audience. Through the publications of 3 journal articles, we expanded our audience to scientists interested in this area or research to the entire world. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
During this period, our research efforts were focused on discovering changes in proteomes in tomato roots that were caused by toxic levels of Aluminum. With the support of Dr. Thannhauser and the use of his laboratory in the Soil and Nutrition Research Unit at the USDA-ARS in Ithaca, NY we were able to identify proteins that were induced/supressed by toxic ions in tomato leaves and roots. Some of these proteins had never been associated previously with ion toxicity. This is a novel discovery by itself. In addition to growing tomato plants in media containing toxic levels of Aluminum, we performed analysis on plants that were grown under salt and heat stress with tolerant wild tomato species and susceptible cultivated tomatoes to identfy unigenes and conserved orthologous sequences that are common or unique to different abiotic stress factors. This information is providing the bases for the isolation of regulatory elements for functional genes, and for the development of tools (molecular markers) that can be used by tomato breeders. Through this project, a formal collaboration between TSU and a USDA laboratory located at Cornell University was established. This collaboration will be used to promote both faculty and students training from minority-serving institutions. We have just been awarded two new research grants(in 2010)that will use the technology gained through the USDA collaboration and additional grants are being applied for. To expand the research scope, we are making over-expression and knock-down transgenic plants that will contain the selected genes and we will evaluate their function to determine if these trangenic plants have increase tolerance to aluminum. The effects of AL accumulation in tomato fruits will also be evaluated. Through this research we will discover genes that could be introduced in other crop plants that will have the ability to survive and florish in soils containing toxic levels of Al.
Publications
- No publications reported this period
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: We have completed cDNA microarray and proteomics analysis. This work was done in order to understand the molecular responses to aluminum toxicity in the tomato. In addition to studies with aluminum, we have completed several studies dealing with salt stress. Through these studies, we have identified several molecular changes that are specific to salt and aluminum stress factors. PARTICIPANTS: Theodore W. Thannhauser2 2Agricultural Research Service; 102A R. W. Holley Center for Agriculture & Health; Cornell University; Ithaca, NY USA; Braden Boone and Levy Shawn.Vanderbilt Microarray Shared Resource, Vanderbilt University,465 21st Avenue South, MRBIII Room 9274, Nashville, TN 37232, USA. TARGET AUDIENCES: academic and scientific research societies PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Research results from this period have provided the foundation for functional genetics study for Al tolerance in tomato plants. The genes identified from genomics and proteomics analysis are being modified in the tomato. We have identified several candidate genes that have the potential to improve plant tolerance to these stress factors. In addition to provide training opportunities for our graduate students in the laboratory, research methods developed in these studies are being incorporated in our graduate courses. The collaborative arrangements that were formed among TSU, the USDA, and Vanderbilt University made it possible for us to achieve our objectives.
Publications
- Zhou, S. Sauve, R. and Thannhauser, T.W. 2009. Aluminum induced proteome changes in tomato cotyledons. Plant Signaling & Behavior 4:8, 1-4 2.
- Zhou,S., Sauve, R., Thannhauser, T.W. 2009. Proteome changes induced by aluminium stress in tomato roots. Journal of Experimental Botany 2009 60(6):1849-1857; doi:10.1093/jxb/erp065
- Zhou, S. and Sauve, R. 2009. Salt induced protein in tomato roots. J. Amer. Soc. Hort. Sci., March 2009; 134: 289 - 294.
- Zhou,S., Sauve, R., Boone, B. and Levy, S. 2008. Identification of genes associated with aluminum toxicity in tomato roots using cDNA microarrays. Plant Stress 2:113-120.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Upon completion of the microarray analysis of Aluminum treated tomato seedlings, we continued to conduct a proteomics analysis in order to identify genes that are regulated at both transcript and protein level. The proteomics analysis was performed in the USDA-ARS Plant, Soil & Nutrition Laboratory/Cornell University under supervision of Dr. Thannhauser, and with the help of other scientists and staff members. Later on, using the same procedures, we have expanded the research to include salt and water stress in order to look for elements that are commonly regulated by abiotic stresses. In addition, this research project has helped to set up the system for microarray and protein analysis at Institute of Agricultural and Environmental Research at Tennessee State University. Other scientists at TSU will use this facility in their research project. During our presentations, we constantly placed emphasis the toxic potential of Aluminum to agricultural production system and human health to raise public awareness of protecting our environment. PARTICIPANTS: Dr. Suping Zhou. PD, Tennessee State University. Dr. Zhou has been trained on the project on proteomics analysis at USDA-ARS Plant, Soil & Nutrition Laboratory/Cornell University, and microarray procedure at Microarray Core Lab, Vanderbilt Univeristy, Dr.Roger Sauve,Co-PD, Tennessee State University. Ms. Braden Boone and Dr. Levy Shawn, Microarray Core, Vanderbilt University, Collaborator Dr. Shu Wei, Agriculture Canada, Collaborator. Dr. Thannhauser, USDA-ARS Plant, Soil & Nutrition Laboratory/Cornell University, Collaborator TARGET AUDIENCES: We made conference and seminar presentations to faculty and students in Agricultural and Biological Sciences. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The proteomics analysis has identified several proteins associated with antioxidant stress, Al-inducible proteins, and others. The cDNA sequences for these proteins have been retrieved from Solanaceae database and used to design primers for quantitative PCR to determine their transcript abundance after Aluminum treatment. Meanwhile, these genes changed at protein level and transcript level (from microarray analysis) are being compiled with their up/down regulation pattern as well as fold changes. We will submit these data to appropriate database once it is complete. In addition, the genes identified will be the candidate genes for further genetic manipulation to modulate this stress factors. This phase of research has accumulated extensive basic genetic information for understanding tomato aluminum toxicity.
Publications
- Zhou, S., Wei, S., Boone, B. and Levy, S. 2007. Microarray analysis of genes affected by salt stress in Tomato. African Journal of Environmental Science and Technology Vol. 1 (2), pp.014-026.
- Zhou, S. 2007.A proteomics study on aluminum toxicity in tomato. Seminar presentation,Institute of Agricultural and Environmental Research, Tennessee State University. http://www.tnstate.edu/iager/core/seminar series.htm
- Zhou, S. 2007. Microarray transcript profiling to identify aluminum toxicity in tomato.The 29th Annual University-Wide Research Symposium, Tennessee State University, Nashville, TN
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Progress 09/01/06 to 08/31/07
Outputs
We have conducted a proteomics analysis on the aluminum treated tomato seedlings. A list of the proteins have been identified. The microarray analysis to identify the changes in the transcript level is underway.
Impacts
This project so far has established the microarray, proteomics analysis procedure for tomatoes. Using the technique, we have treated tomato seedlings with salt stress and identified many salt affected genes and proteins. The proteins identified for aluminum stress will be the candidate genes for further genetic manipulation to modulate this stress factors. As soon as the transcript regulation of these genes are confirmed, constructs will be made for enhancement/ suppression of these genes to test their functions. This phase of research has accumulated the extensive basic genetic information for understanding tomato aluminum toxicity.
Publications
- No publications reported this period
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Progress 09/01/05 to 08/31/06
Outputs
A tomato root culture and aluminum treatment system has been established. Surface sterilized tomato seeds are growing in a liquid culture medium containing AlCl3 for 20d and then the root tissues can be harvested for isolation of protein and RNA. In the case of inoculating 20 seeds in a 250 ml flask, 0.5g root tissues can be harvested; A cDNA differential display procedure has been established. The total RNA samples were extracted from roots in the liquid cultures of Control (+ H2O) and Al-treatment (+AlCl3). The cDNA differential display procedure was performed using the RNA image Kits (GenHunters, Nashville, Tenn.). The cDNA fragments were separated on denature acrylamide gel and detected on a FMBIOIII Image system. A few gene fragments have been cloned and sequenced; A protein extraction procedure has been established for proteomics analysis. Total protein was extracted from root culture using a Tris- based buffer. The protein sample was precipitated in 20% TCA.
After air-dry at 4C in a refrigerator, the protein powder was dissolved in an IEF buffer. The protein solution was loaded onto the IEF strips and single protein molecules were separated using 2-dimensional electrophoresis. After staining with phosphor-protein staining reagents, several phosphorylated protein spots have been detected. These protein spots were isolated and analyzed by mass spectrometry at Columbia University. A few proteins have been identified and confirmed using western blot. To strengthen the research capacity on protein MS analysis, the PD has applied and been accepted into the Proteomic Workshop at Vanderbilt University (Nashville, Tenn.) in August, 2006. The procedure for cDNA microarray hybridization and data analysis has been established. The PD has participated in a workshop on microarray hybridization at Arizona University in April, 2006. She and one graduate student have received training on slides scanning and data analysis at the Microarray Core of
Vanderbilt University (Nashville, Tenn). In our laboratory at Tennessee State University, we have performed microarray analysis using the Tom2 tomato microarray slides to identify Al-induced genes. We have found 60 genes that have some increases in the Al-treated tomato roots. We could not identify genes that are significantly different between control and Al-treatments. We are in the process of optimizing the root treatment and slide hybridization procedure. The research results have been presented at several scientific conferences including the Conference for the 2006 symposium of Association of Research Directors and the 2006 Conference of American Society for Horticultural Science.
Impacts
We have isolated some gene fragments which will be used for cloning the full-length genes. These genes may be potentially used for relieving Al-toxicity in agricultural production. The identification of Al-affected protein accumulation and modification has prepared the base for the next step of research. Protein post-translational modification has been approved a very important mechanism for gene regulation and stress-response. With the identity of the protein being determined, we will be able to search the DNA sequence and try to study the regulation of the related genes at different stages of transcription, translation and protein modification. These efforts may help us to understand the molecular regulation of Al in root development. Two graduate students have been working on these projects for 1-2 years. They are trained in each step of cDNA differential display and cDNA microarray hybridization, as well as data analysis. In our laboratory, these students are able
to use the techniques and knowledge learnt from this project in other research programs. These techniques are widely used today in biotech industry and national security programs. These students will be able to use these techniques and serve the society.
Publications
- Chen, T.and Zhou, S. 2006. cDNA Differential Display of tomato root culture treated with Aluminum.ARD Symposium,Atlanta, GA.
- Chen, T. and Zhou, S. 2006. Genes induced by aluminum toxicicty in tomato roots. Conference of American Society for Horticultural Science, New Orleans, LA.
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