Progress 09/01/20 to 04/04/23
Outputs
Target Audience:The plant biology community Changes/Problems:A graduate student leading the tomato project experienced chronical mental illness. We have worked with the graduate program and university to allow this student time to seek medical treatment through a leave of absence. The delay was unexpected. Another graduate student has joined the project to move the tomato experiments forward. In addition, the PI has moved to University of Mississippi. It took more than half a year for the University of Florida to close out the project in cooperation with the subawardee the Penn State Univ. What opportunities for training and professional development has the project provided?In the Assmann lab, post-doc Dr. Yotam Zait, and research technician Mr. David Arginteanu have worked on this project, collecting guard cell samples and generating stomatal mutant response data. A graduate student, Ms. Megan Sylvia, led our outreach activity at the Penn State Arboretum. How have the results been disseminated to communities of interest?The results have been disseminated to the communities through several ways: Scientific publications and poster presentations: - Xiang, Q.Y., Lott, A.A., Asssmann, S.M., Chen, S. (2020) Advances and perspectives in the metabolomics of stomatal movement and the disease triangle. Plant Science 302, 110697; doi.org/10.1016/j.plantsci.2020.110697 - Jeevaratnam, S., Lin, C., Chen, S. (2020) Effect of sonication on plant stomatal movement. Journal of Undergraduate Research 22, 1-7 (Fall2020). - David, L., Kang, J., Dufresne, D., Zhu, D., Chen, S. (2021) Multi-omics revealed molecular mechanisms underlying guard cell systemic acquired resistance. International Journal of Molecular Sciences 22(1), 191. doi: 10.3390/ijms22010191. - David, L., Kang, J., Nicklay, J., Dufresne, C., Chen, S. (2021) Identification of DIR1-dependant cellular responses required for guard cell systemic acquired resistance. Frontiers in Molecular Bioscience 8, 746523. doi: 10.3389/fmolb.2021.746523. (15 pages) - Kang, J., David, L., Cang, J., Chen, S. (2021) Three-in-one simultaneous extraction of proteins, metabolites and lipids for multi-omics. Frontiers in Genetics 12, 635971. doi: 10.3389/fgene.2021.635971. (11 printed pages) - Li, Y., Zhu, W., Xiang, Q., Kim, J., Dufresne, C., Liu, Y., Li, T., Chen, S. (2023) Creation of a plant metabolite spectral library for untargeted and targeted metabolomics. International Journal of Molecular Sciences (in press). - David, L., Chen, S. (2021) Effect of Systemic Acquired Resistance on Guard Cell Immunity. The 82nd American Society of Plant Biologists Southern Section Annual Conference, virtual zoom, April 16 -18, 2021. - Lin, L.L., Chhajed, S., Mangual, G., Chen, S. (2021) Role of glucosinolates in stomatal immunity against Pseudomonas syringae. University of Florida Undergraduate Research Symposium (virtual), Gainesville, FL, April 16, 2021. - Pourmoghadam, M., David, L., Chen, S. (2021) Effects of systemic immunity on diurnal stomatal movement of Arabidopsis thaliana. University of Florida Undergraduate Research Symposium (virtual), Gainesville, FL, April 16, 2021. - David, L., Kang, J.N., Li, Y., Chen, S. (2021) Simultaneous extraction of proteins, metabolites and lipids for high coverage multi-omics. The ABRF 2021 Annual Meeting Supporting Interdisciplinary Science, March 7-11, 2021. - Chhajed, S., Lin, L.L., Mangual, G., Dufresne, C., Chen, S. (2022) Metabolomics of stomatal immunity in Arabidopsis wild- type and Vam3 mutant. The 70th ASMS Conference on Mass Spectrometry and Allied Topics (ASMS 2022), Minneapolis, MN, USA, June 5-9, 2022. - Kang, J., David, L., Li, Y., Chen, S. (2021) High coverage multi-omics enabled by three-in-one sample extraction and AcquireX data acquisition. Human Proteome Organization (HUPO) Reconnect 2021, virtual conference, November 15 - 19, 2021. - Chhajed, S., Lin, L.L., Mangual, G., Dufresne, C., Chen, S. (2021) Metabolomics and proteomics of guard cells after Pst DC3000 infection. 69th American Society for Mass Spectrometry Annual Conference, Philadelphia, PA, USA, October 31 - November 4, 2021. Conference and seminar presentations: PI Chen has given the following seminars in which results from the project have been discussed. Analysis of Different Epimers of Glycosphingolipid LcGg4 by Ion Mobility Mass Spectrometry, 2022 Mid-South Glycoscience Meeting, Oxford, MI, USA, June 15-17, 2022. Single cell-type omics of guard cell immunity and CO2 response, College of Biological Sciences and Engineering, Beifang University of Nationalities, Ningxia, China, June 9, 2022 (virtual via zoom). Future directions of plant stress resilience research, Plenary forum speaker for MizzouForward, University of Mizzouri, Columbia, MO, USA, April 18-21, 2022. Systems biology and synthetic biology of plant stress resilience, Department of Biology, University of Mississippi, Oxford, USA, March 23-25, 2022. Systems biology of stomatal disease triangle. Institute of Agriculture, University of Tennessee at Knoxville, USA, March 2, 2022. Mass spectrometry technologies for multi-omics biomedical research. International Conference of the Genetics Society of Korea 2021, Seoul National University Siheung Campus, Seoul, South Korea, October 20-21, 2021 The role of melatonin in stomatal disease triangle. Department of Plant Pathology, University of Florida, Gainesville, Florida,USA, October 12, 2021. Single cell-type omics of stomatal disease triangle. Department of Biochemistry, University of Missouri, Columbia, Missouri, USA, October 1, 2021. Tomato stomatal disease triangle. Chinese Science and Technology Forum on Tomato Biology-Basics and Applications, Hosted by Shenyang Agricultural University, Shenyang, China, September 17-19, 2021. - Systems biology of stomatal disease triangle and CO2 response. Department of Biological Sciences at Northern Illinois University, virtual zoom, April 6, 2021. - Single-cell type proteomics and metabolomics of guard cell immunity. 2nd International Conference on Plant Science and Research (virtual zoom conference). Chiang Mai, Thailand, November 2 - 3, 2020. - Single cell-type metabolomics revealed a novel high CO2 signaling pathway. Association of Biomolecular Resource Facilities (ABRF) Education Workshop on Single-Cell 'Omics: Technologies and Applications, via Zoom, October 22, 2020. What do you plan to do during the next reporting period to accomplish the goals?This is the final technical report (FTR) for work at the University of Florida. The funding will be transferred to University of Mississippi. After the fund arrives at the University of Mississippi, here are the work to be done: - Complete sample collection of stomatal guard cells for temporal metabolomics of pathogen-triggered stomatal closing and re-opening - Conduct untargeted and targeted metabolomics to identify new metabolites that play a role in pathogen-triggered stomatal movement - To characterize the functions of candidate metabolites (e.g., melatonin and oxylipin) using reverse genetics, etc. - Continue to train students at different levels, especially minority students in plant metabolomics and stomatal disease triangle. ?
Impacts
What was accomplished under these goals?
This project is a jointly funded and coordinated project between NSF and USDA. This is also a collaborative project between groups at University of Florida (UF) (Dr. Sixue Chen) and Penn State University (PSU) (Dr. Sarah M. Assmann). The goal is to study the roles of metabolites in plant guard cell immunity using both the reference plant Arabidopsis thaliana and tomato. The major activities in this past two years include: 1) Wrote up and published a review on metabolomics of stomatal disease triangle;2) Established a robust method for enriching stomatal guard cells - blender prep. 3) Developed a three-in-one extraction method for isolating metabolites, lipids and proteins from the same sample, enabling multi-omic analyses of the stomatal disease triangle. 4) Created a plant metabolite spectral library for untargeted and targeted metabolomics. It has been publishedin IJMS; 5) Using our robust blender prep method for enriching stomatal guard cells, we have collected control, Pst DC3000 and Pst DC3118 treated guard cells in a time course for targeted and untargeted metabolomics; 6) Using our three-in-one extraction method, we conducted multi-omic analyses of a lipid transfer protein mutant, which has defect in stomatal defense; 7) We started hypothesis-testing experiments using reverse genetics; 8) Our virtual plant metabolomics workshop/symposium has been posted on the YouTube (https://www.youtube.com/playlist?list=PLEUvSo2Avk_-WTZT_1xiJ_NACZv7vi9C1). There are more than 850 views. Specific Objectives: 1) To summarize recent advances in metabolomics of stomatal disease triangle (done during Covid lockdown) 2) To establish a robust blender prep for enriching guard cells. 3) To develop a three-in-one method for multi-omics. 4) To create a plant metabolite spectral library for untargeted/targeted metabolomics 5) To collect guard cell samples and to analyze metabolomic changes in the course of Pst DC3000 and Pst DC3118- triggered stomatal movement 6) To determine DIR1-mediated multi-omic changes in stomatal defense response 7) To characterize signaling and metabolic mutants and their potential functions in stomatal immune responses 8) To publicize an organized national metabolomics workshop and symposium Significant Results: 1. Wrote up and published a review on metabolomics of stomatal disease triangle. As this project started during COVID-19 pandemic, our wet lab experiments at both UF and PSU were interrupted. We used our work-from-home time to summarize current progress on the metabolomics of stomatal movement and the disease triangle. Our review paper was published in Plant Science in October 2020. 2. Established robust methods for enriching stomatal guard cells- blender prep. We developed a robust stomatal guard cell preparation protocol in Arabidopsis and tomato. Through many tests and optimization, our collaborative team has established a blender preparation protocol, which allowed us to enrich stomatal guard cells for metabolomics. 3. Developed a three-in-one sample preparation method for multi-omics of stomatal disease triangle. The standard methanol and isopropanol metabolite extraction method yields mostly hydrophilic metabolites. The other molecules, e.g., lipids and proteins are discarded. Here we developed a three-in-one extraction and fractionation method, where metabolites, lipids and proteins are collected simultaneously from the same sample. As a proof-of-concept, we profiled the molecular changes in guard cells of an Arabidopsis Defective in Induced Resistance 1 (DIR1) mutant versus wild-type (WT). This paper has been published. ?4. To create a plant metabolite spectral library for untargeted/targeted metabolomics.Using the 544 authentic standards that we acquired, we have created a plant metabolite spectral library for both untargeted metabolomics and targeted metabolomics. The spectra were incorporated into a mzVault spectral library. Our mzVault spectral library increased the number of level 1-identified metabolites in a pilot untargeted study. The mzVault library has been deposited in a public repository for free access by the community (deposited in Zenodo, DOI: 10.5281/zenodo.6916522). 5. To analyze metabolomic changes in the course of PstDC3000 and PstDC3118- triggered stomatal movement.We were recently invited to write a detailed protocol for a book chapter in Methods in Enzymology (Chhajed et al., (2022) "A Three-in-One protocol for high throughput plant multiomics". Methods in Enzymology). An important step was to fine tune the blending method to isolate highly purified guard cells at large scale. For each time point, samples are collected for three conditions. These three conditions are: 1) a mock with opening buffer (as defined above); 2) Pseudomonas syringae pv. tomato DC 3000 resuspended to a final O.D. of 0.2 in opening buffer, and; 3) Pseudomonas syringae pv. tomato DC3118 also resuspended in opening buffer to a final O.D. 0.2. To date, ~1,080 leaves have been processed for one complete time course of 0, 30, 60, 90, 120 and 180 mins for each of the 3 conditions. A second time course has been partially completed. Each time point is sampled on a different day, but on each day, observations are made on small subset of stomatal peels subjected to the entire timecourse, to confirm that the stomata are responding in the characteristic manner. 6. To determine DIR1-mediated multi-omic changes in stomatal defense response.The LIPID TRANSFER PROTEIN DEFECTIVE IN INDUCED RESISTANCE 1 (DIR1) mutant is known for its defect in plant defense. Here we demonstrate that stomatal response to pathogens is also altered in the dir1 mutant. We have determined potential signaling mechanisms specific for guard cells by profiling metabolite, lipid, and protein differences between guard cells in the wild type and dir1 mutant. We identified two long-chain 18 C and 22 C fatty acids and two 16 C wax esters as potential targets of DIR1. This work has been published in Frontiers in Molecular Bioscience. 7. To characterize Arabidopsis signaling and metabolic mutants and their functions in stomatal immune responses. We have screened many Arabidopsis signaling and metabolic mutants for their potential stomatal immunity phenotypes (e.g., lox1, wrky18, wrky40, wrky60, npr1, ics1, trxf1, prxII, hak1, sif3, aha1, aha2, gpa1- 3, and ca1ca4). In the last reporting period, we identified an interesting melatonin triple mutant. When treated with Pst DC3118 (coronatine deficient), WT stomata close at 1 hour and remain closed. In the melatonin triple mutant, treatment with Pst DC3000 showed the same result as WT. Surprisingly, after Pst DC3118 treatment, the mutant closed stomata at 1 hour, but reopened stomata at 3 hours. Exogenous supplementation with melatonin (Mel) can reverse the mutant reopening and cause WT to be hyposensitive to the Pst treatment. We are working on hypothesis-testing experiments to elucidate possible interplays between melatonin, oxylipins, jasmonic acid, salicylic acid and coronatine. We will also work on lox1 and gpa1. These hypothesis-testing experiments will help characterize important nodes and edges in the stomatal defense networks. As to the tomato work, we have also started to work on some hormone mutants. The progress has been slow due to chronical mental illness of a graduate student on the project. We have worked with the graduate program and university to allow this student time to seek medical treatment through a leave of absence. Another graduate student has joined the project to move the tomato experiments forward. 8)We organized a virtual national metabolomics workshop and symposium (https://www.youtube.com/playlist?list=PLEUvSo2Avk_-WTZT_1xiJ_NACZv7vi9C1). Therecordings of workshop tutorials and symposium lectures were disseminated on YouTube, and have attracted more than 850 views. There were 555 registrants including 251 women and 36 underrepresented minority attendees (of which 20 were women).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Li, Y., Zhu, W., Xiang, Q., Kim, J., Dufresne, C., Liu, Y., Li, T., Chen, S. (2023) Creation of a plant metabolite spectral library for untargeted and targeted metabolomics. International Journal of Molecular Sciences 24, 2249. https://doi.org/10.3390/ijms24032249 (13 printed pages)
- Type:
Book Chapters
Status:
Published
Year Published:
2022
Citation:
Chhajed, S., Lu, L.L., Mangual, G., Dufresne, C., Chen, S. (2022) Three-in-One method for high throughput plant multiomics. Methods in Enzymology DOI:10.1016/bs.mie.2022.08.039
|
Progress 09/01/21 to 08/31/22
Outputs
Target Audience:The plant biology community Changes/Problems:A graduate student leading the tomato project experiencedchronical mental illness. We have worked with the graduate program and university to allow this student time to seek medical treatment through a leave of absence. The delay was unexpected.Another graduate student has joined the project to move the tomato experiments forward. What opportunities for training and professional development has the project provided?In the Assmann lab, post-doc Dr. Yotam Zait, and research technician Mr. David Arginteanu have worked on this project, collecting guard cell samples and generating stomatal mutant response data. A graduate student, Ms. Megan Sylvia, led our outreach activity at the Penn State Arboretum. How have the results been disseminated to communities of interest?The results have been disseminated to the communities through several ways: 1) Scientific publications and poster presentations: - David, L., Kang, J., Nicklay, J., Dufresne, C.,Chen, S. (2021) Identification of DIR1-dependant cellular responses required for guard cell systemic acquired resistance. Frontiers in Molecular Bioscience 8, 746523. doi: 10.3389/fmolb.2021.746523. (15 pages) - Kang, J., David, L., Cang, J.,Chen, S. (2021) Three-in-one simultaneous extraction of proteins, metabolites and lipids for multi-omics. Frontiers in Genetics 12, 635971. doi: 10.3389/fgene.2021.635971. (11 printed pages) - Chhajed, S., Lin, L.L., Mangual, G., Dufresne, C., Chen, S. (2022) Metabolomics of stomatal immunity in Arabidopsis wild-type andVam3mutant. The 70th ASMS Conference on Mass Spectrometry and Allied Topics (ASMS 2022), Minneapolis, MN, USA, June 5-9, 2022. - Kang, J., David, L., Li, Y., Chen, S. (2021) High coverage multi-omics enabled by three-in-one sample extraction and AcquireX data acquisition. Human Proteome Organization (HUPO) Reconnect 2021, virtual conference, November 15 - 19, 2021. - Chhajed, S., Lin, L.L., Mangual, G., Dufresne, C., Chen, S. (2021) Metabolomics and proteomics of guard cells afterPstDC3000 infection. 69th American Society for Mass Spectrometry Annual Conference, Philadelphia, PA, USA, October 31 - November 4, 2021. 2) Conference and seminar presentations: PI Chen has given the following seminars in which results from the project have been discussed. - Analysis of Different Epimers of Glycosphingolipid LcGg4 by Ion Mobility MassSpectrometry, 2022 Mid-South Glycoscience Meeting, Oxford, MI, USA, June 15-17, 2022. - Single cell-type omics of guard cell immunity and CO2 response, College of Biological Sciences and Engineering, Beifang University of Nationalities, Ningxia, China, June 9, 2022 (virtual via zoom). - Future directions of plant stress resilience research, Plenary forum speaker for MizzouForward, University of Mizzouri, Columbia, MO, USA, April 18-21, 2022. - Systems biology and synthetic biology of plant stress resilience, Department of Biology, University of Mississippi, Oxford, USA, March 23-25, 2022. - Systems biology of stomatal disease triangle. Institute of Agriculture, University of Tennessee at Knoxville, USA, March 2, 2022. - Mass spectrometry technologies for multi-omics biomedical research. International Conference of the Genetics Society of Korea 2021, Seoul National University Siheung Campus, Seoul, South Korea, October 20-21, 2021 - The role of melatonin in stomatal disease triangle. Department of Plant Pathology, University of Florida, Gainesville, Florida, USA, October 12, 2021. - Single cell-type omics of stomatal disease triangle. Department of Biochemistry, University of Missouri, Columbia, Missouri, USA, October 1, 2021. - Tomato stomatal disease triangle. Chinese Science and Technology Forum on Tomato Biology-Basics and Applications, Hosted by Shenyang Agricultural University, Shenyang, China, September 17-19, 2021. What do you plan to do during the next reporting period to accomplish the goals?- Complete sample collection of stomatal guard cells for temporal metabolomics of pathogen-triggered stomatal closing and re-opening -Conduct untargeted and targeted metabolomics to identify new metabolites that play a role in pathogen-triggered stomatal movement -To characterize the functions of candidate metabolites (e.g., melatonin and oxylipin) using reverse genetics, etc. -Continue to train students at different levels, especially minority students in plant metabolomics and stomatal disease triangle.
Impacts
What was accomplished under these goals?
This project is a jointly funded and coordinated project between NSF and USDA. This is also a collaborative project between groups at University of Florida (UF) (Dr. Sixue Chen) and Penn State University (PSU) (Dr. Sarah M. Assmann). The goalis to study the roles of metabolites in plant guard cell immunity using both the reference plantArabidopsis thalianaand tomato. The major activities in this past year include: 1) Created a plant metabolite spectral library for untargeted and targeted metabolomics. This library will not only be used for this project, but also will be a great community resource; 2) Using our robust blender prep method for enriching stomatal guard cells, we have collected control,PstDC3000 andPstDC3118 treated guard cells in a time course for targeted and untargeted metabolomics; 3) Using our three-in-one extraction method for isolating metabolites, lipids and proteins, we conducted multi-omic analyses of a lipid transfer protein mutant, which has defect in stomatal defense; 4) Westarted hypothesis-testing experiments using reverse genetics. We have screened many signaling and metabolic mutants for stomatal immunity phenotypes, and prioritized them for detailed functional characterization studies; 5) Ourvirtual plant metabolomics workshop/symposiumhas beenposted on theYouTube(https://www.youtube.com/playlist?list=PLEUvSo2Avk_-WTZT_1xiJ_NACZv7vi9C1). There are more than 850 views. Specific Objectives: The specific objectives for the past year included: 1) To create a plant metabolite spectral library for untargeted/targeted metabolomics 2) To collect guard cell samples and to analyze metabolomic changes in the course ofPstDC3000 andPstDC3118-triggered stomatal movement 3) To determine DIR1-mediated multi-omic changes in stomatal defense response 4) To characterize signaling and metabolic mutants and their potential functions in stomatal immune responses 5)To publicize an organizednational metabolomics workshop and symposium Significant Results: 1.To create a plant metabolite spectral library for untargeted/targeted metabolomics.The lack of plant authentic standards and metabolite spectral library limit the identification of plant metabolomics study. Using the 544 authentic standards that we acquired, we have created a plant metabolite spectral library for both untargeted metabolomics and targeted metabolomics. The spectra were incorporated into a mzVault spectral library. Our mzVault spectral library increased the number of level 1-identified metabolites in a pilot untargeted study. The mzVault library has beendeposited in a public repository for free access by the community (deposited inZenodo,DOI: 10.5281/zenodo.6916522). Furthermore, based on the spectral library, we developed a process of creating targeted metabolomics methods without the need of purchasing authentic standards. In the future, more metabolite spectra can be easily incorporated into the library to improve the mzVault package. In addition, more metabolites can be profiled by designing sensitive and selective targeted metabolomics methods using the mzVault spectral library. 2.To collect guard cell samples and to analyze metabolomic changes in the course of Pst DC3000 and Pst DC3118-triggered stomatal movement. We have published our three-in-one extraction and fractionation method in Frontiers in Genetics. It collects metabolites, lipids and proteins simultaneously from the same sample, allowing for conservation of experimental materials, such as guard cells, and for multi-omics studies. We were recently invited to write a detailed protocol for a book chapter in Methods in Enzymology (Chhajed et al., (2022) "A Three-in-One protocol for high throughput plant multiomics". Methods in Enzymology). An important step was to fine tune the blending method to isolate highly purified guard cells at large scale.For each time point, samples are collected for three conditions. These three conditions are: 1) a mock with opening buffer (as defined above); 2)Pseudomonas syringae pv.tomato DC 3000 resuspended to a final O.D. of 0.2 in opening buffer, and; 3)Pseudomonas syringae pv.tomato DC3118 also resuspended in opening buffer to a final O.D. 0.2. To date, ~1,080 leaves have been processed for one complete time course of 0, 30, 60, 90, 120 and 180 mins for each of the 3 conditions. A second time course has been partially completed. Each time point is sampled on a different day, but on each day, observations are made on small subset of stomatal peels subjected to the entire timecourse, to confirm that the stomata are responding in the characteristic manner. Once the samples are ready, we will use the spectral libraries and conduct untargeted and targeted metabolomics to analyze metabolomic changes in the course ofPstDC3000 andPstDC3118-triggered stomatal movement. 3.To determine DIR1-mediated multi-omic changes in stomatal defense response.TheLIPID TRANSFER PROTEIN DEFECTIVE IN INDUCED RESISTANCE 1 (DIR1)mutant is known for its defect in plant defense. Here we demonstrate that stomatal response to pathogens is also altered in the dir1 mutant. Using a multi-omics approach, we havedetermined potential signaling mechanisms specific for guard cells by profiling metabolite, lipid, and protein differences between guard cells in the wild type anddir1mutant. We identified two long-chain 18 C and 22 C fatty acids and two 16 C wax esters as potential targets of DIR1. This work has been published in Frontiers in Molecular Bioscience. 4.To characterize Arabidopsis signaling and metabolic mutants and their potential functions in stomatal immune responses.In addition todir1, we have screened many Arabidopsis signaling and metabolic mutants for their potential stomatal immunity phenotypes (e.g.,lox1, wrky18, wrky40, wrky60, npr1, ics1, trxf1, prxII, hak1, sif3, aha1, aha2, gpa1-3,andca1ca4). In the last reporting period, we identified an interesting melatonin triple mutant. When treated withPstDC3118 (coronatine deficient), WT stomata close at 1 hour and remain closed. In the melatonin triple mutant, treatment withPstDC3000 showed the same result as WT. Surprisingly, afterPstDC3118 treatment, the mutant closed stomata at 1 hour, but reopened stomata at 3 hours. Exogenous supplementation with melatonin (Mel) can reverse the mutant reopening and cause WT to be hyposensitive to thePsttreatment. We are working on hypothesis-testing experiments to elucidate possible interplays between melatonin, oxylipins, jasmonic acid, salicylic acid and coronatine. We will also work onlox1andgpa1. These hypothesis-testing experiments will help characterize important nodes and edges in the stomatal defense networks. As to the tomato work, we have also started to work on some hormone mutants. The progress has been slow due to chronical mental illness of a graduate student on the project. We have worked with the graduate program and university to allow this student time to seek medical treatment through a leave of absence. Another graduate student has joined the project to move the tomato experiments forward. 5.To publicize a national metabolomics workshop and symposium.Weorganized a virtual national metabolomics workshop and symposium (https://www.youtube.com/playlist?list=PLEUvSo2Avk_-WTZT_1xiJ_NACZv7vi9C1). The recordings of workshop tutorials and symposium lectures were disseminated on YouTube, and have attracted more than 850views. There were 555 registrants including 251 women and 36 underrepresented minority attendees (of which 20 were women). In addition, Sixue Chen has co-organized a National Science Foundation sponsored national posttranscriptional and posttranslational modifications workshop on October 18-20th, 2021 (https://biotech.ufl.edu/event/nsf-sponsored-workshop-cross-disciplinary-study-of-post-transcriptional-and-post-translational -modifications/). Dr.Assmann gave a talk, participated in the discussions, and provided comments on the resulting white paper.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
David, L., Kang, J., Nicklay, J., Dufresne, C., Chen, S. (2021) Identification of DIR1-dependant cellular responses required for guard cell systemic acquired resistance. Frontiers in Molecular Bioscience 8, 746523. doi: 10.3389/fmolb.2021.746523. (15 pages)
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Kang, J., David, L., Cang, J., Chen, S. (2021) Three-in-one simultaneous extraction of proteins, metabolites and lipids for multi-omics. Frontiers in Genetics 12, 635971. doi: 10.3389/fgene.2021.635971. (11 printed pages)
- Type:
Book Chapters
Status:
Submitted
Year Published:
2022
Citation:
Chhajed, S., Lu, L.L., Mangual, G., Dufresne, C., Chen, S. (2022) A Three-in-One protocol for high throughput plant multiomics. Methods in Enzymology (invited)
|
Progress 09/01/20 to 08/31/21
Outputs
Target Audience:The plant biology community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?To date, two graduate students, Lisa David and Qingyuan Xiang, and a visiting scholar Yangyang Li have received training on metabolomics, mass spectrometry, stomatal physiology and immunity in the Chen lab. A minority undergraduates Soren Jeevaratnam, as well as two women undergraduate students Lin Lu and Marral Pourmoghadam have received training in stomatal movement assays, mutant screening and in metabolite sample preparation. Another undergraduate student in Chemistry Daniel Dufresne received training in mass spectrometry and data analysis. Mr. Soren Jeevaratnam has published his results in Journal of Undergraduate Research. Ms. Marral Pourmoghadam has submitted her work to the Journal of Undergraduate Research. In addition, Dr. Chen and Dr. Assmann are organizing a virtual metabolomics workshop. In the Assmann lab, the project is carried on by an experienced stomatal physiology post-doc, Dr. Yotam Zait, and by 50% time of a research technician, Mr. David Arginteanu. How have the results been disseminated to communities of interest?1) Scientific publications and poster presentations: - Xiang, Q.Y., Lott, A.A., Asssmann, S.M., Chen, S. (2020) Advances and perspectives in the metabolomics of stomatal movement and the disease triangle. Plant Science 302, 110697; doi.org/10.1016/j.plantsci.2020.110697 - Jeevaratnam, S., Lin, C., Chen, S. (2020) Effect of sonication on plant stomatal movement. Journal of Undergraduate Research 22, 1-7 (Fall2020). - David, L., Kang, J., Dufresne, D., Zhu, D., Chen, S. (2021) Multi-omics revealed molecular mechanisms underlying guard cell systemic acquired resistance. International Journal of Molecular Sciences 22(1), 191. doi: 10.3390/ijms22010191. - David, L., Chen, S. (2021) Effect of Systemic Acquired Resistance on Guard Cell Immunity. The 82nd American Society of Plant Biologists Southern Section Annual Conference, virtual zoom, April 16 -18, 2021. - Lin, L.L., Chhajed, S., Mangual, G., Chen, S. (2021) Role of glucosinolates in stomatal immunity against Pseudomonas syringae. University of Florida Undergraduate Research Symposium (virtual), Gainesville, FL, April 16, 2021. - Pourmoghadam, M., David, L., Chen, S. (2021) Effects of systemic immunity on diurnal stomatal movement of Arabidopsis thaliana. University of Florida Undergraduate Research Symposium (virtual), Gainesville, FL, April 16, 2021. - David, L., Kang, J.N., Li, Y., Chen, S. (2021) Simultaneous extraction of proteins, metabolites and lipids for high coverage multi-omics. The ABRF 2021 Annual Meeting Supporting Interdisciplinary Science, March 7-11, 2021. 2) Conference and seminar presentations: PI Chen has given the following seminars in which results from the project have been discussed. - Systems biology of stomatal disease triangle and CO2 response. Department of Biological Sciences at Northern Illinois University, virtual zoom, April 6, 2021. - Single-cell type proteomics and metabolomics of guard cell immunity. 2nd International Conference on Plant Science and Research (virtual zoom conference). Chiang Mai, Thailand, November 2 - 3, 2020. - Single cell-type metabolomics revealed a novel high CO2 signaling pathway. Association of Biomolecular Resource Facilities (ABRF) Education Workshop on Single-Cell 'Omics: Technologies and Applications, via Zoom, October 22, 2020. What do you plan to do during the next reporting period to accomplish the goals? - Collect stomatal guard cells for temporal metabolomics of pathogen-triggered stomatal closing and re-opening - Identify new metabolites that play a role in pathogen-triggered stomatal movement - Functional studies assessing candidate metabolites using reverse genetics, etc. - Organize a virtual metabolomics training workshop in 2021, and an in-person hands-on training workshop in spring or summer of 2022.
Impacts
What was accomplished under these goals?
Major Activities: This is an interagency (NSF and USDA), jointly funded and coordinated project. We have decided to focus on Arabidopsis (a reference plant) for the NSF support and tomato (an agriculturally important crop) for the USDA support. This is also a collaborative project between groups at University of Florida (Dr. Sixue Chen) and Penn State University (Dr. Sarah M. Assmann). The grant was designed to facilitate research into the roles of metabolites in plant guard cell defense. In this first year of funding, COVID19 has caused many difficulties. Our groups did our best to meet these challenges and make good use of the time: we worked on literature review, experimental design and strategies, and continued to be productive. The major activities in this past year include: 1) Wrote up and published a review on metabolomics of stomatal disease triangle. 2) Established a robust method for enriching stomatal guard cells - blender prep. We summarized current progress on the metabolomics of stomatal movement and the disease triangle. The manuscript was published in Plant Science in October 2020; 3) Developed a three-in-one extraction method for isolating metabolites, lipids and proteins from the same sample, enabling multi-omic analyses of the stomatal disease triangle. 4) Expanded the analysis of the guard cell metabolome from about 400 metabolites to more than 800 metabolites. In addition to three-dimensional analysis of the metabolome (retention time, mass to charge, and peak area), we have added a fourth dimension of ion mobility; 5) Started to characterize new metabolites that play a role in pathogen-triggered stomatal movement, e.g., the roles of melatonin and lipid-related metabolites in stomatal movement and defense. We have also tested several mutants for altered guard cell defense, including a zinc-binding dehydrogenase (At3g45770) in fatty acid degradation, a sphingosine kinase (At5g23450) and a phospholipase D alpha 1 (At3g15730). Specific Objectives: The specific objectives for the past year included: 1) To write up the literature on the stomatal disease triangle 2) To establish a robust stomatal guard cell preparation protocol. 3) To develop a three-in-one sample preparation method for multi-omics of stomatal disease triangle. 4) To expand the analysis of the guard cell metabolome by establishing a comprehensive metabolite spectral library for targeted and untargeted metabolomics. 5) To characterize the roles of newly identified metabolites in stomatal movement and defense using reverse genetics Significant Results: 1. Wrote up and published a review on metabolomics of stomatal disease triangle. As this project started in the middle of the COVID-19 pandemic, our wet lab experiments at both UF and PSU were interrupted and required dedensification of personnel at PSU is an ongoing issue. In addition, we also experienced personnel recruitment problem. In spite of the challenges, in the early days of the pandemic, we used our work-from-home time to summarize current progress on the metabolomics of stomatal movement and the disease triangle. Our review paper was published in Plant Science in October 2020. 2. Established robust methods for enriching stomatal guard cells- blender prep. Despite our limited time in the laboratory, we succeeded to develop a robust stomatal guard cell preparation protocol in Arabidopsis and tomato. The protocol that we developed before used transparent Scotch tape. While it works well for proteomics, it is problematic for metabolite and lipid extraction due to increased volume and plastic contamination from the tape. Through many tests and optimization, our collaborative team has established a blender preparation protocol, which does not use tape. The obtained stomatal guard cells are intact and viable, and the prepared guard cells are highly enriched. 3. Developed a three-in-one sample preparation method for multi-omics of stomatal disease triangle. The standard methanol and isopropanol metabolite extraction method yields mostly hydrophilic metabolites. The other molecules, e.g., lipids and proteins are discarded. Here we developed a three-in-one extraction and fractionation method, where metabolites, lipids and proteins are collected simultaneously from the same sample. This three-in-one sample preparation method allows for conservation of experimental materials, such as guard cells, and for multi-omics studies. It is going to be very useful for this project. 4. Expanded the analysis of the guard cell metabolome by establishing a comprehensive metabolite spectral library. Structural annotation of metabolites is a grand challenge of metabolomics. The best way to meet this challenge is to acquire authentic chemical standards for generating experimental spectral libraries, which can be used for both targeted and untargeted metabolomics. Based on the biochemical pathway database for Arabidopsis (AraCyc), we acquired 544 authentic standards last year. This year we were able to increase the number to more than 800 standard compounds.In addition to three-dimensional analysis of the metabolome (retention time, mass to charge, and peak area), we have added a fourth dimension of ion mobility. We are currently in the middle of building a four-dimensional spectral library in Metaboscape software. The spectral library can be used to generate targeted metabolomics methods, such as multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM), and can also be used for metabolite identification from untargeted metabolomics data. It will be not only important for expanding the analysis of the guard cell metabolome in this project, but also will provide a critical resource for the plant metabolomics community. 5. Reverse genetics characterization of the roles of newly identified metabolites in stomatal movement and defense. Based on preliminary results of metabolite changes in guard cells, we have tested several mutants in jasmonic acid (JA) synthesis and signaling for altered guard cell immune responses. In addition, we have also assessed mutants of a zinc-binding dehydrogenase (At3g45770) in fatty acid degradation, a sphingosine kinase (At5g23450) and a phospholipase D alpha 1 (At3g15730). One of the aims of the stomatal disease triangle is to determine effects of CO2 on stomatal immunity. In our previous NSF support, we have characterized stomatal movements and guard cell metabolomic responses to high and low CO2 concentrations. Under low CO2 condition, we observed a significant melatonin increase in the early stage of stomatal opening. The exact opposite change, i.e., decrease of melatonin, was found under high CO2. Melatonin is a newly discovered plant hormone. Its role in stomatal immunity is not known. Here we compared the stomatal movements of wild-type (WT) and melatonin triple mutant in response to Pst DC3000 and Pst DC3118.WT stomata close at 1 hour and reopen at 3 hours after Pst DC3000 treatment. The reopening is due to secretion of coronatine by the Pst DC3000. When treated with Pst DC3118 (coronatine deficient), WT stomata close at 1 hour and remain closed. In the melatonin triple mutant, treatment with Pst DC3000 showed the same result as WT. Surprisingly, after Pst DC3118 treatment, the mutant closed stomata at 1 hour, but reopened stomata at 3 hours. Exogenous supplementation with melatonin (Mel) can reverse the mutant reopening and cause WT to be hyposensitive to the Pst treatment. Based on the current knowledge, we came up with a model about possible interplays between Mel, SA, JA-Ile and coronatine. We are currently working on hypothesis-testing experiments. Additionally, we have acquired different tomato mutants and WT lines. Testing their stomatal immune responses is under way.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
David, Lisa and Kang, Jianing and Dufresne, Daniel and Zhu, Dan and Chen, Sixue. (2021). Multi-Omics Revealed Molecular Mechanisms Underlying Guard Cell Systemic Acquired Resistance. International Journal of Molecular Sciences. 22 (1) 191.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Xiang, Qingyuan and Lott, Aneirin A. and Assmann, Sarah M. and Chen, Sixue. (2021). Advances and perspectives in the metabolomics of stomatal movement and the disease triangle. Plant Science. 302 (C) 110697.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Jeevaratnam, S., Lin, C., Chen, S. (2020). Effect of sonication on plant stomatal movement. Journal of undergraduate research. 22.
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