Source: UNIV OF NORTH CAROLINA submitted to NRP
REGULATION OF RICE GROWTH AND YIELD BY THE CYTOKININ TYPE-A RESPONSE REGULATORS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030800
Grant No.
2023-67012-40242
Cumulative Award Amt.
$225,000.00
Proposal No.
2022-09620
Multistate No.
(N/A)
Project Start Date
May 15, 2023
Project End Date
May 14, 2026
Grant Year
2023
Program Code
[A1152]- Physiology of Agricultural Plants
Recipient Organization
UNIV OF NORTH CAROLINA
(N/A)
CHAPEL HILL,NC 27514
Performing Department
(N/A)
Non Technical Summary
As the global population continues to grow, so do our needs for resources and food. The cereal crop rice provides ~30% of the calories consumed by humans globally. Increasing rice robustness and yield (number of grains) would help to meet these global food needs. Previous work has demonstrated that higher levels of the plant hormone cytokinin positively influence grain production in rice, specifically by increasing the number of grains produced by each panicle (inflorescence). This is a very promising lead to follow, as manipulation of this process would produce high-yield rice. However, the regulatory mechanism behind this phenomenon is not yet understood. By using the popular gene editing system CRISPR/Cas9, we have developed an extensive collection of rice lines with precise edits disrupting a group of genes (called the type-A Response Regulators) known to inhibit cytokinin response--meaning these plants should have heighted response to cytokinin and increased grain yield. Further, by using traditional breeding techniques, we are crossing these lines to wild type plants and select those individuals that retain the precise mutation(s) of interest but lack transgenic material. We are studying these lines closely to identify plants with better performance by measuring important traits such as plant size, number of days to flower, number of panicles/inflorescences per plant, ability to efficiently harvest light energy (photosynthesis), and grain yield. Using these data, we will identify the most productive lines and study gene expression via RNA-sequencing in their inflorescence meristems--the developmental tissue that gives rise to the mature panicle and, ultimately, grains--as compared to wild type (un-edited) plants. From these data, genes targeted by the type-A RRs will be identified and studied further; thus, shedding light on the mechanism of type-A RR action. Overall, this work will bring us much closer to establishing highly productive field-ready rice lines.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20115301050100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1530 - Rice;

Field Of Science
1050 - Developmental biology;
Goals / Objectives
The primary focus of this work is to assess the roles of the type-A RRs (players in the cytokinin signaling pathway in plants) in various aspects of plant development, including grain yield, individual grain size, fitness, and the rate of photodamage. The direct outcomes of this work will be to identify rice lines with alterations to grain yield and rates of photosynthesis as compared to the wild type and identify specific downstream genes determining these traits regulated by the type-A RRs from transcriptional data in wild type and mutant lines. The proposed research will elucidate the role of the type-A RRs in regulating gene expression and growth and development in rice, furthering our understanding of the downstream targets and mechanisms of this signaling pathway.Objectives:(1) Finalize multiple knockout mutants in rice for the four type-A RR clades.CRISPR/Cas9-generated multiple gene knockout lines (by clade) for the type-A RRs (negative regulators of the cytokinin signaling pathway) will be finalized. These lines will be genotyped by sequencing and checked for presence of the CRISPR/Cas9 transgene via PCR screening. Where possible, secondary alleles and lower-order mutants within each clade will be identified and screened in the same manner. Lines lacking the CRISPR/Cas9 transgene will be isolated for downstream experiments.(2) Characterize plant morphology and productivity in the four type-A rr lines.Using the type-A rr mutant lines lines generated in (1), growth and yield-relevant traits will be measured at various plant growth stages in order to identify the effects of disruption of these genes on grain yield. A large-scale greenhouse morphological screen will be carried out to gather data such as tiller number, floral morphology, panicle number, and grain yield in these lines. Seedling assays will also be conducted to characterize seedling morphology, and cytokinin response assays will be done to measure sensitivity to this hormone in the lines. The data generated from these experiments will be used to identify possible clade-specific regulated traits and guide future experiments. This work will also determine if any of these lines exhibit increased grain yield.(3) Characterize gene expression patterns and identify probable transcriptional targets of the type-A RRs in developing rice panicles.Transcriptome sequencing will be done with developing panicles (inflorescences) from the most promising mutant line identified in (2) (i.e., the line with the greatest increase in yield) and wild type plants. Differential gene expression analysis will be conducted to (a) characterize broad changes in the pattern of gene expression between wild type and the mutant, and (b) identify genes targeted by the type-A RRs. Together with the data generated in (2), a better mechanism for the roles of the type-A RRs in cytokinin signaling in rice will be established.
Project Methods
EffortsThe aims of this project are: 1) finalize multiple knockout mutants in rice for the four type-A RR clades, 2) characterize plant morphology and productivity in the four type-A rr lines, and 3) identify identify probable transcriptional targets of the type-A RRs in the developing rice panicle. Knockout rice lines for the type-A RRs are being made using CRISPR/Cas9, and mutations identified through a combination of sanger sequencing and PCR. Back crosses (to wild type) are being done with established mutant lines to remove the transgenic CRISPR/Cas9 cassette, producing stable non-transgenic lines. Plant morphology and productivity will be assessed from a large-scale screen carried out in the green house. Many traits will be measured--grain yield will be determined by counting the number of filled seeds/panicle in matured plants, and photosynthesis traits measured using a portable spectrophotometer device, to name a couple. Developing panicles will be isolated from the most promising mutant line identified in this screen (e.g., the line with the greatest increase in yield as compared to wild type) and wild type, RNA extractions done, and RNAseq performed to generate transcriptome data. These data will be analyzed following a well-established differential expression analysis pipeline to identify key genes of interest that are expressed differentially in wild type versus type-A rr during panicle development. EvaluationThe direct outcomes of this work include a collection of stable non-transgenic type-A rr knockout rice lines, measurements of important agricultural traits in these lines, and isolation of lines that are more productive compared to wild type (i.e., with increased grain yield and with altered rates of photosynthesis). Transcriptome data will be generated from the developing panicles of wild type and the most interesting type-A rr line identified, and a list of genes potentially targeted by the type-A RRs produced. Research findings are anticipated to be published as 1-2 journal articles and presented at at least 2 conferences in either the form of posters or talks. A public GitHub repository will be established for all code and data analysis methods associated with the project. At least 2 undergraduate students will be recruited to work on the project (as mentees to the PD) and present their findings in papers and/or posters each semester. The broader significance of the project and its potential societal contributions will be conveyed to the public at the UNC Science Expo each April.

Progress 05/15/24 to 05/14/25

Outputs
Target Audience:The scientific/academic community: The PD presented a poster on this project at the Plant Biology 2024 conference (through the American Society of Plant Biologists) and gave a talk on the work in the fall of 2024 at the 35th annual Plant Molecular Biology Retreat. The PD has also routinely presented her progress on this project with the lab and a wider group of collaborators with updates and when seeking advice. Undergraduate students: The PD served as a direct mentor to 1 undergraduate student (working directly on this project) and helped to mentor 2 graduate students in the lab this year. The undergraduate student , having since completed a research proposal to the Biology department, is helping to complete experiments for the project and will continue to work with the PD over the summer and through the next academic year. While the 2 graduate students were not working solely/directly on this project, they utilized materials generated from it and contributed relevant data. Mentorship consisted in the PD assisting the students in learning and troubleshooting techniques and primarily working on their understanding and presentation of their work. The PD has gained value experience in mentoring, especially in how to proceed and adjust with the needs and level of the student. The PD gave a guest lecture on light signaling in plants in her mentor's higher-level seminar course at UNC. This was valuable teaching experience for the PD, as she selected the subject, materials, and created the lecture format. The PD also spent some time after class to answer any questions the students had about graduate school and academic research, specifically what the career is like. General public: The PD provided input, materials, and participated in the lab's UNC Science Expo booth at UNC in spring 2025, where children and adults attended to interact with scientists, talk, and ask any questions. The PD's booth exhibited several plant species, which were used to demonstrate what plant research looks like and why it is important. Changes/Problems:Several building/maintenance issues came up during the year (greenhouse light replacement and glass roof cleaning were delayed, which could have influenced experimental results), which affected the schedule of certain experiments. While finalization of one of the mutant lines were a major factor in delaying the large-scale morphological experiment, these technical issues contributed as well. The other change is our shift in focus on generating gene expression data from developing panicles to leaves. While we believe this change will be much more applicable towards addressing yield, it does complicate sample collection, as we are having to determine the timepoints, types of tissue to collect, as well as treatments to include. However, given our 1-year extension, we firmly believe this work can be completed in this time period. What opportunities for training and professional development has the project provided?The PD gained valuable experience in mentoring and teaching. She is currently mentoring a sophomore undergraduate students, who will assist with the project over the summer and through the year. This has been a very hands-on experience for both, as the student did not have any lab experience before. The PD also worked with 2 graduate students in the lab, helping them with a variety of things, from working with rice to statistical and computational work. The PD was able to attend job talk seminars for a new faculty position at UNC. The experience of meeting with and seeing the candidates present has been invaluable, and helped the PD to start thinking about what she needs to prepare in order to take that next step in the coming years. The writing and maintenance of this project with the USDA is something she will keep in mind when applying for early-career funding. The PD also attended two professional development meetings/events, which introduced her to new people in the plant research field, in addition to learning what other labs and groups (across academia, industry, and government) are doing. Attending the USDA meeting in summer 2024 was particularly enlightening, and meeting the other PDs present was a wonder opportunity to make connections. How have the results been disseminated to communities of interest?The PD presented a poster on this project at the Plant Biology 2024 conference (through the American Society of Plant Biologists) and gave a talk on the work in the fall of 2024 at the 35th annual Plant Molecular Biology Retreat. The PD has also routinely presented her progress on this project with the lab and a wider group of collaborators with updates and when seeking advice. What do you plan to do during the next reporting period to accomplish the goals?This summer will be devoted to (1) completing pilot experiments and protocols for the source/sink experiment and (2) starting the final large-scale plant morphology experiment with all mutants. Results for both will be gathered during the fall. The PD routinely works on an outline for the manuscript this work will go into and will begin writing it up as results start to come in early-fall, with the goal being to submit it for publication before May 2025.

Impacts
What was accomplished under these goals? (1)Finalize multiple knockout mutants in rice for the four type-A RR clades. Generation of the CRISPR/Cas9 multiple gene knockout lines (by clade) for the type-A RRs was completed and seed stocks bulked for following experiments. These lines were thoroughly genotyped and found to be stable, clean lines. Secondary alleles and lower-order mutants were isolated for a subset of the genes. Crosses will be repeated so as to obtain more lower-order lines in case they are needed. (2) Characterize plant morphology and productivity in the fourtype-A rrlines. First, it was found that a typical cytokinin sensitivity/response assay used in the lab (by measuring root growth) did not work with these type-A rr lines. A new approach was identified and protocol developed and tested. We have since shown that the type-A rr lines are indeed more sensitive to cytokinin, in that they grow shoots much faster than normal plants when treated with cytokinin. Next, an interesting observation was made about yield and leaf senescence in one of the higher-order type-A rr lines. We found that the some of the rr lines make more spikelets/florets (future grains) than normal plants. However, the majority of these spikelets never fill with starch (never become grains). It was then found that more of these spikelets would fill and become grains if part of the leaf was cut off the plant. This led us to believe that there is a source/sink issue in these rr lines, and we have adjusted experiments to better test this and see if grain production can be recovered in this line. We have found that this rr line is resistant to senescence, the natural process by which grains would fill, and that these plants have more chlorophyll than normal plants. (3) Characterize gene expression patterns and identify probable transcriptional targets of the type-A RRs in developing rice panicles. We have held off on generating gene expression data as originally planned (from developing panicles), and for good reason. The original purpose was to use this data to identify downstream links between the type-A rrs and how grain yield is regulated via these genes. However, given our findings in (2) about increased spikelet number but lower yield, we believe transcriptome data from leaves over the course of plant senescence/grain filling would be much more useful regarding increasing yield. We are now running pilot experiments to best figure out how to do this.

Publications


    Progress 05/15/23 to 05/14/24

    Outputs
    Target Audience: The scientific/academic community The PD presented a poster on this project atthe Plant Biology 2023 conference (through the American Society of Plant Biologists). The PD has also routinely presented her progress on this project with the lab and a wider group of collaborators with updates and when seeking advice. Undergraduate students The PD helped in mentoring 3 undergraduates in the lab this year. While these students were not working solely/directly on the project, they utilized materials generated from it and contributedrelevant data. Mentorship consisted in the PD assisting the students in learning and troubleshooting techniquesand primarily working on their understanding and presentation of their work. The PD gave a guest lecture dedicated to signaling in plants in the course of her mentor. This was valuable teaching experience for the PD, as she worked to create the lecture format, materials, work with the students and address their questions, and design exam questions. The PD also brought some of her work into the spotlight during the lecture, highlighting this work in an effort to convey the importantce of plant research and related career opportunities to the students, most of which were seniors beginning to decide on their career paths. General public The PD provided input and materials towards the lab's UNC Science Expo booth (she was unable to attent due to a scheduling conflict). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PD has been able to work with her mentor on several professional development activities: Giving a guest lecture in the senior-level signaling course taught by Dr. Kieber Serving as a co-reviewer for Nature Communications for one manuscript (with Dr. Kieber) Continuing to refine writing and presentation skills in the design posters and continuous 'storytelling' development practices through lab/collaborator meeting presentationsand discussions on manuscript outlines Attending several faculty candidate job talks at UNC-CH How have the results been disseminated to communities of interest?Results were presented at Plant Biology 2023 (by ASPB). An undergraduate working on the project (mentored by the PD) completed his senior honors thesis in 2023, resulting in a written thesis as well as a seminar presentation. The PD will present a poster at Plant Biology 2024 this summer. What do you plan to do during the next reporting period to accomplish the goals?Now that mutant lines have been settled and pilot experiments conducted, final runs of these experiments will be completed. This should be finished by Fall 2024. The PD has begun outlining a manuscript for this work.

    Impacts
    What was accomplished under these goals? Below each objective (1-3) I have listed what has been accomplished. (1)Finalize multiple knockout mutants in rice for the four type-A RR clades. CRISPR/Cas9-generated multiple gene knockout lines (by clade) for the type-A RRs (negative regulators of the cytokinin signaling pathway) have beenfinalized. These lines have been genotyped by sequencing and checked for presence of the CRISPR/Cas9 transgene via PCR screening in addition to growth on the selective antibiotic. Backcrosses to WT were performed for 3/4 of the clades and lower-order mutants are being identified currently. Secondary alleles have been identified for some genes, and we are continuously screening for these. (2) Characterize plant morphology and productivity in the fourtype-A rrlines. A PILOT run has been completed for the experiment "A large-scale greenhouse morphological screen will be carried out to gather data such as tiller number, floral morphology, panicle number, and grain yield in these lines." The experiment will be repeated this summer. Preliminary results indicate that loss of type-A RR function, particularly in the RR3,5,6,7,11 clade, results in increased numbers of spikelets and secondary branches. Seedling assays were conducted to measure cytokinin sensitivity to this hormone, ultimately leading to adjustments in the experimental procedure to better characterize hypersensitivityin the lines. Callus, shoot, and root induction assays were conducted with rr3,5,6,7,11, which was found to regenerate shoots faster than WT. Photosynthesis and chlorophyll content data was collected for one line thus far. This assay has been established and will be re-employed to all lines during the summer morphological experiment. (3) Characterize gene expression patterns and identify probable transcriptional targets of the type-A RRs in developing rice panicles. Transcriptome sequencing was done with developing panicles (inflorescences) from the most promising mutant line identified thus far. Differential gene expression analysis revealed few results. The experiment is being repeated with additional mutant lines and the analysis pipeline adjusted for higher-sensitivity.

    Publications