Progress 01/15/24 to 01/14/25
Outputs
Target Audience:The audience for this work includes students studying mechanisms of plant genetics and biochemistry. There were six students working on the project in 2024 for 8-12 hours a week during the spring and fall semesters, and two students worked over the summer (1 full time and 1 part time) for additional hours. The other audience for this work is other scientists who want to modify maize for resilience to stress. I discussed this work with colleagues at the American Society of Plant Biology annual conference, in June 2024. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Six students were directly trained and two were indirectly trained by this project through the group discussions. These are undergraduate research students who work part-time over the academic semesters and full- or part-time over the summer. The PD works directly with the students to teach them how to plan, perform, and analyze experiments related to the project. The students learn how to carry out the experiments on their own for replicates. In addition to teaching the students lab techniques, I train them in leadership, communication, planning, time-management, and organization that contribute to their long-term success as scientifically trained professionals. Two students presented this work at both the Northeast American Society of Plant Biologists regional conference (April 2024) and at the national ASPB conference in June 2024. They wrote senior papers and are contributing to the manuscript for this work. How have the results been disseminated to communities of interest?This work was discussed in my courses at The College of New Jersey. The students are general biology undergraduates with little exposure to the field of plant biology. I taught them to analyze research data and helped them understand the role of plant biology research in our society. It has also been presented at regional and national plant biology conferences. What do you plan to do during the next reporting period to accomplish the goals?We still need to analyze the double mutants for altered stress responses in maize. We are working on a publication for the GA-inactivating maize enzymes, with results from the single mutant as well as overexpression mutants. The work will be presented at regional and national plant biology conferences, and one undergraduate will be working on the project over the summer of 2025.
Impacts
What was accomplished under these goals?
We are studying the mechanisms for maize acclimation to environmental stresses, such as heat, chilling, drought, and combinations of these stresses with caterpillar attack. Objective 1: Isolate additional CYP72A mutations and alter expression levels with virus constructs. We completed the permitting to obtain CRISPR-Cas9 mutants for CYP72A28 and a double mutant in CYP72A28 and CYP72A349. Wisconsin Crop Innovation Center at the University of Wisconsin, Madison, sent us the seeds to begin testing at TCNJ. In the absence of additional mutations in maize, we obtained overexpression mutants with four of the maize genes in Arabidopsis. Objective 2: Measure abiotic and biotic stress responses associated the CYP72A mutations. We designed additional stress experiments, acclimation to multiple iterations of stress and heat stress earlier in the seedling life. The CYP72A overexpression mutants are showing root growth, anthocyanin variation, and resilience to accumulating reactive oxygen species. Objective 3: Determine the chemical nature of metabolites produced by CYP72As. We cloned CYP72A genes into an overexpression vector for making transgenic Arabidopsis thaliana. This work is to directly test the hypothesis that one of the maize genes has the GA-inactivation activity demonstrated in CYP72A9 from Arabidopsis. We found that three of the maize genes have GA-inactivating activity and one does not. We are further measuring reporter genes for other pathways that are altered by the overexpression of CYP72A genes from corn.
Publications
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Progress 01/15/23 to 01/14/24
Outputs
Target Audience:This work impacted the education of undergraduate students and a technician in my research lab at The College of New Jersey. There were five students working on the project in the spring 2023 semester for 8-12 hours a week as their independent research in the Biology Program. Over the summer, two of the students worked full time and one worked part time on the project. The research technician, hired in 2022, worked for 24 hours per week towards this project. In the fall 2023 semester, three students work 10-12 hours a week on this project as part of their research coursework. The project also supports the education of two other students that work in my research lab. I discussed this work with colleagues at the American Society of Plant Biology annual conference, in Savannah GA, in August 2023. The poster related to this work was entitled, "Exploring the Role of CYP72A Enzymes in Zea mays Stress Responses". Changes/Problems:We requested a second no-cost extension due to the delays in the first year in getting into the lab and training students. We are finishing the mutant analysis and working on disseminating the results through presentations at American Society of Plant Biologists meetings and through publications. What opportunities for training and professional development has the project provided?Five students were directly trained and two were indirectly trained by this project through the group discussions. These are undergraduate research students who work part-time over the academic semesters and full- or part-time over the summer. The PD works directly with the students to teach them how to plan, perform, and analyze experiments related to the project. The students learn how to carry out the experiments on their own for replicates. In addition to teaching the students lab techniques, I train them in leadership, communication, planning, time-management, and organization that contribute to their long-term success as scientifically trained professionals. The research technician continues to work on the objectives of this grant. He is learning lab techniques and professional development similar to the undergraduates, but his responsibilities are greater. He has more opportunity to learn to design new experiments, troubleshoot and manage the lab resources. He is also building his leadership skills in mentoring students in the lab and lab management skills for taking care of the overall needs of the lab. How have the results been disseminated to communities of interest?This work was discussed in my Plant Genetics class at The College of New Jersey. The students are general biology undergraduates with little exposure to the field of plant biology. I taught them to analyze research data and helped them understand the role of plant biology research in our society. What do you plan to do during the next reporting period to accomplish the goals?The full complement of personnel is working on this project with multiple students and the research technician. There are two seniors contributing to the analysis of GA-inactivating activity. They are working towards a publication for this work before they go on to graduate school in plant biology. The VIGS and CRISPR mutants will be additional tools to support the hypotheses we are testing with the Arabidopsis over-expression mutants. These tools will help us determine the contributions of several genes to the stress response processes. We will also use the variation in gene expression levels in maize in-bred lines to supplement our mutant collection.?
Impacts
What was accomplished under these goals?
1) Isolate additional CYP72A mutations and alter expression levels with virus constructs 1. We made anti-sense Virus-induced gene silencing vectors for CYP72A28 and CYP72A349. We are in the process of making transgenic plants with gene knockdown. We have demonstrated that the genes are knocked down, and are working to determine whether the knockdown is consistent in single and double mutants. 2. We completed the contracting agreements to initiated CRISPR-Cas9 mutant production for CYP72A28 and a double mutant in CYP72A28 and CYP72A349. The vectors are complete and the mutations are in the corn mutant pipeline at the Wisconsin Crop Innovation Center at the University of Wisconsin, Madison. We have not yet obtained the single and douple mutant seeds. 3. We examined gene expression levels for additional genes to determine which other CYP72A genes are the best targets for additional mutation. In this process, we also determined that splicing is an additional component of transcriptional regulation. The mature RNA is sometimes decreased in abundance due to intron retention. 2) Measure abiotic and biotic stress responses associated the CYP72A mutations 1. There appears to be a mild growth-related phenotype to the stress, suggesting that plants without the CYPs are not acclimating to the stress as well as normal plants. Caterpillars appeared to prefer plant missing CYP72A349 plants to the wildtype when the plants are first subjected to cold stress, suggesting that the normal function of CYP72A349 deters caterpillar feeding. 2. We also performed gene expression analysis on CYP72As from three different corn varieties to determine whether there was natural variation in the contribution each gene makes to the stress response. We have found variants in genetic responses to stress, suggesting that the recombinant in-bred lines could be useful alternatives to mutants for some of the stresses. We continue to explore the degree of gene expression difference in the recombinant inbred lines. There appears to be a difference in intron retention in W22 and B73 wild type plants. 3. The outcome of this work is new knowledge about the role of CYP72A enzymes in plant stress responses. 3) Determine the chemical nature of metabolites produced by CYP72As 1. We cloned CYP72A genes into an overexpression vector for making transgenic Arabidopsis thaliana. This work is to directly test the hypothesis that one of the corn genes has the GA-inactivation activity demonstrated in CYP72A9 from Arabidopsis. 2. We are measuring more GA inactivation in the Arabidopsis plants expressing CYP72A349 from corn. 3. We also measured metabolites in mutant and WT plants exposed to cold stress and caterpillars. We are currently processing the metabolite data.
Publications
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience: This work impacted the education of undergraduate students and a technician in my research lab at The College of New Jersey. There were three students working on the project in the spring 2021 semester for 8-10 hours a week as their independent research in the Biology Program. Over the summer, two of the students worked full time and I hired a research technician for 29 hours per seek towards this project. A third summer student indirectly benefited from the scientific discussions and data analysis that we carried out as a group. In the fall 2022 semester, two students work 10-12 hours a week on this project as part of their research coursework. The project also supports the education of two other students that work in my research lab. I discussed this work with colleagues at the American Society of Plant Biology annual conference, in Portland, OR, in July 2021. My poster was entitled, "Characterizing the role of CYP72A enzymes in maize acclimation to combinations of environmental stresses". Changes/Problems:We requested a no-cost extension due to the delays in the first year in getting into the lab and training students. There was additional delay in getting the CRISPR-Cas9 mutants generated, because the original vender (Iowa State University) closed for outside plant transformation projects. We were able to find another source (Wisconsin Crop Innovation Center at the University of Wisconsin, Madison). The mutant plants are in the pipeline, but we are not likely to have seeds for extensive analysis by the end of the grant period.We requested a no-cost extension due to the delays in the first year in getting into the lab and training students. There was additional delay in getting the CRISPR-Cas9 mutants generated, because the original vender (Iowa State University) closed for outside plant transformation projects. We were able to find another source (Wisconsin Crop Innovation Center at the University of Wisconsin, Madison). The mutant plants are in the pipeline, but we are not likely to have seeds for extensive analysis by the end of the grant period. What opportunities for training and professional development has the project provided?Three students were directly trained and four were indirectly trained by this project through the group discussions. These are undergraduate research students who work part-time over the academic semesters and full- or part-time over the summer. The PD works directly with the student to teach them how to plan, perform, and analyze experiments related to the project. The students learn how to carry out the experiments on their own for replicates. In addition to teaching the student lab techniques, I train them in leadership, communication, planning, time-management, and organization that contribute to their long-term success as scientifically trained professionals. I also hired a research technician who is a recent graduate of our Biology Program. He is learning lab techniques and professional development similar to the undergraduates, but his responsibilities are greater. He has more opportunity to learn to design new experiments, troubleshoot and manage the lab resources. He is also building his leadership skills in mentoring students in the lab. How have the results been disseminated to communities of interest?I discussed this work with colleagues at the American Society of Plant Biology annual conference, in Portland, OR, in July 2021. My poster was entitled, "Characterizing the role of CYP72A enzymes in maize acclimation to combinations of environmental stresses". What do you plan to do during the next reporting period to accomplish the goals?The full complement of personnel is working on this project with multiple students and the research technician. I am seeking working to make sure that there are two full-time students working in the lab this summer in addition to the technician. We will screen plants with CYP72A genes silenced and we should get our CRISPR mutants by the end of the year. These tools will help us determine the contributions of several genes to the stress response processes. We will also use the variation in gene expression levels in maize in-bred lines to supplement our mutant collection. We will compare growth and insect defense responses in plant subjected to a variety of stresses. We plan to travel to the lab of the collaborator to complete metabolite analysis on plant tissue that we have collected in the last two years.?
Impacts
What was accomplished under these goals?
Impact In the second year of this project, the major impacts have been on educating students on research techniques for plant responses to environmental stress, generating additional mutant tools, and collecting data on plant stress responses. We measured maize plant growth under heat and salt stress to determine that one of the CYP72A genes appears to be helping the plants acclimate to the stress in the early weeks of growth. We measured growth responses in other combinations of stresses. We compared caterpillar growth on mutant and normal plants that were first exposed to stress. We collected tissue from plants under a variety of stresses for metabolite analysis. In addition to impacting the education of students in the process of science related to this project, we are making progress toward identifying new mechanisms that plants respond to environmental stressed. With ever changing climate conditions making it difficult to obtain good crop yields, our work is contributing to the understanding of how plants naturally acclimate to stresses. The work will help us better understand which metabolic responses are most critical for optimizing plant performance. What was accomplished toward specific objectives? 1) Isolate additional CYP72A mutations and alter expression levels with virus constructs 1. We made anti-sense Virus-induced gene silencing vectors for CYP72A28 and CYP72A349. We are in the process of making transgenic plants with gene knockdown. 2. We completed the contracting agreements to initiated CRISPR-Cas9 mutant production for CYP72A28 and a double mutant in CYP72A28 and CYP72A349. The vectors are complete and the mutations are in the corn mutant pipeline at the Wisconsin Crop Innovation Center at the University of Wisconsin, Madison. 3. We examined gene expression levels for three additional genes to determine which other CYP72A genes are the best targets for additional mutation. 2) Measure abiotic and biotic stress responses associated the CYP72A mutations 1. We measured abiotic stress responses in 10-day old maize plants exposed to heat, salt, heat+salt, cold, drought, and caterpillar feeding after salt stress. We compared the growth of plants missing CYP72A349 with normal plants. We also did phenotyping for plants missing CYP72A26. 2. There appears to be a mild growth-related phenotype to the stress, suggesting that plants without the CYPs are not acclimating to the stress as well as normal plants. The caterpillars appeared to prefer normal plants to the mutants, suggesting that the metabolic profile of the mutant plants under stress was altering the interactions between the plant and the caterpillar. 3. We also performed gene expression analysis on CYP72As from three different corn varieties to determine whether there was natural variation in the contribution each gene makes to the stress response. We have found variants in genetic responses to stress, suggesting that the recombinant in-bred lines could be useful alternatives to mutants for some of the stresses. 4. The outcome of this work is new knowledge about the role of CYP72A enzymes in plant stress responses. 3) Determine the chemical nature of metabolites produced by CYP72As 1. We cloned CYP72A349 into a cyanobacterial expression system to test the hypothesis that this maize enzyme is able to inactivate gibberellin (GA) growth hormones with the same mechanism as a homolog in Arabidopsis thaliana. We could not detect GA inactivation from the maize enzyme. 2. This work suggests that CYP72A9 is not a growth hormone modulator and is impacting the stress response in a different way.
Publications
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:This work impacted the education of undergraduate students in my research lab at The College of New Jersey. There were two students working on the project in the spring 2021 semester at a low level as we got the lab going again from the pandemic closure. Over the summer, one student worked full time and one student worked part time on this project. A third student indirectly benefited from the scientific discussions and data analysis that we carried out as a group. In the fall 2021 semester, three students work 5-8 hours a week on this project as part of their research coursework. The project also supports the education of two other students that work in my research lab. I discussed this work informally with colleagues at the American Society of Plant Biology annual conference, held virtually in July 2021. I was able to get feedback on our progress. Changes/Problems:Due to restrictions on access to the lab and travel because of the pandemic, we are delayed in expending the research funds as laid out in the project timeline. For the first three months of the award, we were limited in accessing the lab. Only one student was allowed to work in the lab for a few hours a week. Another student was able to work on computational analysis remotely. We did get students into the lab over the summer, but the numbers were still limited, so hiring the research technician was delayed. Because of the significant delays in personnel access to the research lab, we will be delayed by a year in performing many of the research objectives. I will submit a request for an extension on the grant. What opportunities for training and professional development has the project provided?Five students were directly trained and two were indirectly trained by this project through the group discussions. These are undergraduate research students who work part-time over the academic semesters and full- or part-time over the summer. The PD works directly with the student to teach them how to plan, perform, and analyze experiments related to the project. The students learn how to carry out the experiments on their own for replicates. How have the results been disseminated to communities of interest?This work was discussed in my Plant Genetics class at The College of New Jersey. The students are general biology undergraduates with little exposure to the field of plant biology. I taught them to analyze research data and helped them understand the role of plant biology research in our society. What do you plan to do during the next reporting period to accomplish the goals?I am in the process of hiring a research technician to enhance our ability to move the project forward. With an additional experienced research assistant, we should be able to isolate new single and double mutants for research objective 1. We will continue to analyze plant growth under combinations of stresses for research objective 2. As we analyze plants, we will collect them for metabolite comparisons. Objective 3 is dependent on reduced travel restrictions and access to the instruments in our collaborator's institution. We were not able to use travel support or all of the personnel funding for the first year of this project because of the restrictions on hiring and travel due to the pandemic. We are moving forward to reinstate these objectives when allowed.
Impacts
What was accomplished under these goals?
Impact: In the first year of this project, the major impacts have been on educating students on research techniques for plant responses to environmental stress and collecting data on the second research objective. We measured maize plant growth under heat and salt stress to determine that one of the CYP72A genes appears to be helping the plants acclimate to the stress in the early weeks of growth. We contributed new knowledge on the role these enzymes play in plant stress responses. Specific objectives: 1) Isolate additional CYP72A mutations and alter expression levels with virus constructs We completed the Materials Transfer Agreement paperwork to begin using a vector for Virus-Induced Gene Silencing to make a double mutant of two CYP72A genes that are closely related. We do not have data, results, or outcomes for the new mutants. 2) Measure abiotic and biotic stress responses associated the CYP72A mutations We measured abiotic stress responses in 10-day old maize plants exposed to heat, salt or heat+salt. We compared the growth of plants missing CYP72A349 with normal plants. We focused on this one gene to test the hypothesis that young plants were more sensitive to the stress. We measured plant height and shoot weight for plants exposed to each stress. The plants were germinated under normal conditions and exposed to stress for two days. The mutant plants (missing CYP72A349) were significantly larger than the wild-type plants, suggesting that the absence of this enzyme prevents them from slowing their growth in response to the stress. Interestingly, the combination of heat and salt stress reduced the growth for both mutant and wild-type plants. This result supports our hypothesis that there is another gene responsible for modulating plant growth in response to these stresses. The outcome of this work is new knowledge about the role of CYP72A enzymes in plant stress responses. CYP72A349 appears to help regulate the growth/defense tradeoff as young corn plants acclimate to abiotic stress. 3) Determine the chemical nature of metabolites produced by CYP72As We examined the substrate binding pocket of CYP72A349 in computational models. We were not able to directly test metabolites due to travel restrictions. The models were tested for substrate interactions with plant hormones to test the hypothesis that these enzymes are involved in the growth-defense tradeoff. There was no significant evidence that the growth hormones fit in the binding pocket of CYP72A349 in a configuration that would support the predicted inactivation. The outcome of this work is inconclusive because there are limitations in the computational structural analysis. We need to directly measure the metabolites.
Publications
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