Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The target audience is the scientific community that seeks to understand genetic regulatory mechanisms controlling developmental processes and mechanisms of transcription factor function. Specifically, we are interested in seed development and somatic embryogenesis. To this end, we published two manuscripts during the review period. A dataset was publishied in NCBI GEO reporting on targets of ABI3 (superseries accession GSE150561). One Ph.D. student completed his degree. Both graduate students and the postdoctoral scholar presented their reserarch as poster presentations at the Plant Biology 2019 meeting. We have also been involved in outreach activities to the general public to inform and interest them in plant biology, and the relevance of seeds to society. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this most recent reporting period, one Agricultural and Medical Biotechnology (ABT) student worked in the lab. She is a non-traditional student who transferred from a community college, is a bit older and is in her 30's, than her colleagues and has a new baby. She first acquired experience during spring 2019, and then as well as experience, credit toward her degree in fall 2019. She is an excellent worker who will appear on a future publication from the lab. During the fall semester, a postdoctoral scholar has been primarily responsible for very effectively mentoring this student in techniques of interest to the ABT student, namely site-directed mutagenesis and cloning. A second ABT student was involved in the lab in Spring 2020. Unfortunately, COVID-19 meant that she was unable to participate after March 2020 due to restrictions on undergraduates in the lab. The lab has also hosted for the third year a high school student from the Carter G. Woodson Academy (CGWA). CGWA works with underrepresented male minority students who are "at risk" academically. The student with whom my lab has worked is now a senior. This program consists of 99% minority male students. He had a tendency to miss school, at least in part due to late night work schedules. This has resolved since he has been involved in the lab. He is brilliant! He understands concepts introduced in the lab beyond what he has learned in high school, and can reproducibly perform pretty complex experimental procedures, for example, qRT-PCR. He won first place in Plant Sciences at the regional science affair. This was very significant because he was or still is, very nervous presenting in front of an audience. We were a good combination because I was able to share with him that I too feel nervous but it does get better with time. Clearly, he overcame this to effectively present his work to the judges. Some of our planned work was disrupted by COVID-19 and he has now graduated from high school. He is currently serving in the National Guard and beginning his college work at the community college. His long term goal is to work as a veterinarian or in agricultural science. Two Ph.D. students are involved in the project. One completed his work this summer and has now moved to his postdoctoral position in Texas. He was the first author on two papers, as well as preparing and defending his dissertation. The other graduate student and the postdoc coauthored a paper this year. Both have manuscripts under preparation. The Senior lab technician has supported the lab in a large number of ways. She also was involved in developing an outreach program to a local elementary school and training people to assist her with running this workshop. She was awarded a College Outstanding Staff Award this fall. How have the results been disseminated to communities of interest?Two manuscripts have been published. One Ph.D. dissertation was successfully defended. Three posters were presented at the ASPB Plant Biology 2019 meeting. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. Verify in planta AGL15-protein interactions. We have confirmed co-immunoprecipitation from plant tissues for several proteins first identified as interacting with AGL15 by yeast 2-hybrid. These include a related and at least partially redundantly acting MADS AGL18, and a transcription factor in the LOB domain family. 2. Determine in vivo targets of AGL15 interacting proteins. We have now acquired RNA-seq data to examine the transcript accumulation in response to SAP18 in seedlings and developing seeds. We tested these stages to determine how the transcriptome may change during this important developmental phase transition. We have also assessed the transcriptome in response to LBD40, AGL18 and HDA19. To allow for better comparison, we also assessed the transcriptome in response to AGL15 to complement and expand on prior microarray results. RNA-seq does not identify targets that are necessarily directly regulated by these factors, so ChIP seq has also been performed for these factors. We are currently analyzing data to determine direct vs. indirect targets, as well as examining overlap of directly regulated networks. During this review cycle, one graduate student wrote and successfully defended his dissertation. We expect an additional manuscript about SAP18 from this work. In addition, weare actively working on two manuscripts reporting on results from AGL18 and LBD40. 3. Investigate mechanisms by which AGL15 can directly express and repress transcription. Thus far, the loop experiments have not been reproducibly positive. Constructs have been made to mutate AGL15 binding sites in the 5' and 3' ends of FLC to which AGL15 binds and further experiments will be underway. Less progress on this aim has been make as we have been focusing more on aim 2. However, we have looked at effects of different regions of AGL15 on gene expression versus repression, and post-translational modifications with some intriguing results. AGL18 has also been part of this analysis. A third manuscript is actively under preparation reporting on these results. 4. Further investigate the GmAGL15 associated gene regulatory network in soybean. We have performed RNA-seq for overexpression of GmAGL15 (that promotes somatic embryogenesis) in the explants (0 day after culture, dac) and 3 and 7 dac. This expands upon what we learned with microarrays where less than half the gene are represented. We are, in the analysis, getting an overlapping but also different picture of what happens during early somatic embryo development. The goal was to also perform ChIP-seq for AGL15 - this has been difficult because the transgenic we were using is not tagged with an epitope and we were relying on cross-reactivity of anti-AGL15 sera raised against a Brassica napus AGL15 protein to cross-react with the soybean protein. While this has been productive for testing select genes, acquiring a robust enough population for ChIP-seq has been questionable. Therefore, we have been testing soybean transgenic lines that have a 10x-c-myc tag for which we have a tested, commercially available c-myc antibody that works for ChIP. We are testing lines for ChIP-seq for GmAGL15 and GmAgl18 and for RNA-seq for GmAGL18. We have been having difficulties seeing reproducible protein accumulation and this part of the project has been de-emphasized due to COVID-19 and restrictions on people in the lab.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Ran Tian, Fangfang Wang, Qiaolin Zheng, Venus Niza, A. Bruce Downie and Sharyn E. Perry (2020) Direct and indirect targets of the Arabidopsis seed transcription factor ABSCISIC ACID INSENSITIVE3. The Plant Journal 103: 1679-1694
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Ran Tian, Priyanka Paul, Sanjay Joshi and Sharyn E. Perry (2020) Genetic activity during early plant embryogenesis. Biochemical Journal 447: 3743-3767
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2020
Citation:
Ran Tian (2020) Regulatory networks during seed development. July 15, 2020
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audience is the scientific community that seeks to understand genetic regularoty mechanisms controlling developmental processes and mechanisms of transcription factor function. Specifically, we are interested in seed development and somatic embryogenesis. We have also been involved in outreach activities to the general public to informand interest them in plant biology, and the relevance of seeds to society. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this most recent reporting period, one Agricultural and Medical Biotechnology student worked in the lab. She is a non-traditional student who transferred from a community college, is a bit older and is in her 30's, than her colleagues and has a new baby. She first acquired experience during spring 2019, and then as well as experience, credit toward her degree in fall 2019. She is an excellent worker who will appear on a future publication from the lab. During the fall semester, a postdoctoral scholar has been primarily responsible for very effectively mentoring this student in techniques of interest to the ABT student, namely site-directed mutagenesis and cloning. The lab has also hosted for the third year a high school student from the Carter G. Woodson Academy. CGWA works with underrepresented male minority students who are "at risk" academically. The student with whom my lab has worked is now a senior. This program consists of 99% minority male students. He had a tendency to miss school, at least in part due to late night work schedules. This has resolved since he has been involved in the lab. He is brilliant! He understands concepts introduced in the lab beyond what he has learned in high school, and can reproducibly perform pretty complex experimental procedures, for example, qRT-PCR. He won first place in Plant Sciences at the regional science affair. This was very significant because he was or still is, very nervous presenting in front of an audience. We were a good combination because I was able to share with him that I too feel nervous but it does get better with time. Clearly, he overcame this to effectively present his work to the judges. He thinks he would like to be a veterinarian and I am currently working with him on college applications and mentoring from those in vet sciences. Two Ph.D. students are involved in the project. One is a co-author on a paper in revision. The progress described above under "accomplishments" are the result of their and a postdocs hard work. We expect a number of manuscripts within the next year or two. How have the results been disseminated to communities of interest?Three lab members (two graduate students and one postdoc) attended the Plant Biology meeting in 2019 in San Jose, CA to present posters on their results. All, as well as the PI and technician, have been involved in presentations of our work to the public via workshops, judging at science fairs, and science nights in our community. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. Verify in planta AGL15-protein interactions. We have found that AGL15 can interact with the related AGL18 protein in planta via co-immunoprecipitation experiments. At the last report (2018), we noted a technical difficulty for co-immunoprecipitation with LBD40. LBD40 was identified by yeast 2-hybrid as a potential interacting protein for AGL15 and was especially interesting due to the gene's seed specific expression and co-regulation by many key embryo transcription factors. We have resolved the technical problem (involving detection of the heavy chain of IgG that interfered with detection of LBD40) and confirmed in plant co-immunoprecipitation of AGL15-LBD40. Other interacting proteins are being pursued by the lab members. Given the challenges for co-immunoprecipitation, I am excited that two proteins have been verified. 2. Determine in vivo targets of AGL15 interacting proteins. We have now acquired RNA-seq data to examine the transcript accumulation in response to SAP18 in seedlings and developing seeds. We tested these stages to determine how the transcriptome may change during this important developmental phase transition. We have also assessed the transcriptome in response to LBD40 and AGL18. To allow for better comparison, we also assessed the transcriptome in response to AGL15 to complement and expand on prior microarray results. RNA-seq does not identify targets that are necessarily directly regulated by these factors, so ChIP seq has also been performed for these factors. We are currently analyzing data to determine direct vs. indirect targets, as well as examining overlap of directly regulated networks. 3. Investigate mechanisms by which AGL15 can directly express and repress transcription. Thus far, the loop experiments have not been reproducibly positive. Constructs have been made to mutate AGL15 binding sites in the 5' and 3' ends of FLC to which AGL15 binds and further experiments will be underway. Less progress on this aim has been make as we have been focusing more on aim 2. 4. Further investigate the GmAGL15 associated gene regulatory network in soybean. We have performed RNA-seq for overexpression of GmAGL15 (that promotes somatic embryogenesis) in the explants (0 day after culture, dac) and 3 and 7 dac. This expands upon what we learned with microarrays where less than half the gene are represented. We are, in the analysis, getting an overlapping but also different picture of what happens during early somatic embryo development. The goal was to also perform ChIP-seq for AGL15 - this has been difficult because the transgenic we were using is not tagged with an epitope and we were relying on cross-reactivity of anti-AGL15 sera raised against a Brassica napus AGL15 protein to cross-react with the soybean protein. While this has been productive for testing select genes, acquiring a robust enough population for ChIP-seq has been questionable. Therefore, we have been testing soybean transgenic lines that have a 10x-c-myc tag for which we have a tested, commercially available c-myc antibody that works for ChIP. We are testing lines for ChIP-seq for GmAGL15 and GmAgl18 and for RNA-seq for GmAGL18. As above, we can combine this data to determine direct and indirect targets of AGL15/18 in soybean and Arabidopsis and find the most relevant targets for promotion of somatic embryogenesis.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audience is the scientific community that seeks to understand genetic regularoty mechanisms controlling developmental processes and mechanisms of transcription factor function. Specifically, we are interested in seed development and somatic embryogenesis. We have also been involved in outreach activities to the general public to inform and interest them in plant biology, and the relevance of seeds to society. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the reporting period, two Agricultural and Medical Biotechnology (ABT) undergraduate students received training in lab techniques including safety, sterile technique, PCR (both quantitative and semi-quantitative), running gels, and other techniques. They both are better prepared for their required ABT independent study projects based on the time spent in my lab. In addition, I have been hosting a high school student who is also an underrepresented minority and in a special program for "at risk" students. This program consists of 99% minority and 100% male students. His teacher credits the program with his improved attendance and the student noted it motivated him to pass math. He has been involved in sterile culture, PCR, RNA extractions and quantitative RT-PCR and produced some very nice data. Two Ph.D. students have been involved during this reporting cycle, one of whom is a co-first author on a paper currently under revision. This student served as a Teaching Assistant for a lab-based ABT course in Fall 2017 and Spring 2018 semesters. The other student began his Ph.D. work in 2017 and has now passed his qualifying exams. A postdoctoral scholar also joined the lab Dec. 1, 2017. All have been involved in meeting the goals of the project, training others in the lab, and in outreach activities; one of which was initiated by the senior lab technician. How have the results been disseminated to communities of interest?One paper is currently in revision for resubmission. Upon acceptance the datasets will be deposited at GEO. Members of the lab have been involved in a number of outreach activities including displays on seeds at science fairs (2), hands-on science activities at elementary schools (3), judging at science fairs, and University biotech days (2). The latest outreach to an elementary school involved 120 third graders and 108 fourth graders and taught them some basics of genetics and included DNA extractions. The workshop was very well received and was spear-headed by the senior lab tech in my group and involved all lab members. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. Verify in planta AGL15-protein interactions. - We are still confirming most of the proteins found to interact with AGL15 in yeast 2-hybrid. Tools for co-IP experiments have been developed for all the proteins of interest, and BiFC clones generated to test some interactions. AGL18, the closest relative of AGL15 in Arabidopsis, was found to co-immunoprecipitate with AGL15. 2. Determine in vivo targets of AGL15 interacting proteins. - Tools for chromatin immunoprecipitation (ChIP) have been developed for putative AGL15 interacting proteins. We currently have the first biological replicate for three interacting proteins submitted for NGS by ChIP-seq. As well, we have included ChIP-seq for AGL15 to allow a more direct comparison than the ChIP-chip study in hand. We have also started generating tissue for RNA-seq studies to assess the transcriptome in response to increased and decreased interactor compared to wild type control. We have the first biological replicate for one interacting protein. The additional biological replicates to assess the transcriptome in developing seeds and seedlings in response to this factor have been submitted for NGS. 3. Investigate mechanisms by which AGL15 can directly express and repress transcription - As part of this aim, we have been looking further at the function of different motifs within the C-terminal domain of AGL15. Specifically we have looked at a potential repression motif and assessed the necessity of this motif for promoting somatic embryo development. We are also assessing effects on gene expression. A high school student has been involved in this work and presented a poster at the science fair in February 2018. We have been developing tools for timecourses and competition ChIP. We are generating lines with potential AGL15 target sites mutated to test whether activation of some targets requires two sites. 4. Further investigate the GmAGL15 associated gene regulatory network in soybean. - RNA-seq has been performed on soybean explants overexpressing GmAGL15 compared to control during a timecourse of somatic embryo induction. We are currently assessing additional transgenic lines of GmAGL15 and GmAGL18 to allow comparison.
Publications
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