Source: The Regents of University of California submitted to NRP
INCREASING FOOD PRODUCTION AND NUTRITION THROUGH FERTIGATION AND MIXOTROPHIC GROWTH IN CONTROLLED ENVIRONMENT AGRICULTURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1028197
Grant No.
2022-67011-36632
Cumulative Award Amt.
$180,000.00
Proposal No.
2021-09403
Multistate No.
(N/A)
Project Start Date
Jan 3, 2022
Project End Date
Jan 2, 2025
Grant Year
2022
Program Code
[A7101]- AFRI Predoctoral Fellowships
Recipient Organization
The Regents of University of California
200 University Office Building
Riverside,CA 92521
Performing Department
Chemical and Environmental Eng
Non Technical Summary
Food demand is growing globally, however food production is ultimately constrained by the amount of arable land on the planet. Conventional agriculture is also resource intensive and has an enormous impact on the environment. To continue to feed our growing population, it is essential to increase agricultural yields.Controlled Environmental Agriculture (CEA), in which crops are grown in a controlled hydroponic indoor environment with vertically stacked grow areas, has the potential to produce more food with fewer resources in a sustainable way. However, CEA is very energy intensive and expensive. The electricity needed to run the grow lights for CEA is its largest energetic and financial cost.This research aims to reduce the cost of CEA and increase its productivity through fertigation and mixotrophic growth. The controlled nature of CEA provides opportunities to use fertigation, a method of providing plants with nutrients through their irrigation system. Plants grown in CEA could be fertigated with non-standard nutrient mixes such as fermented wastewater or even given an additional chemical energy source to increase food production, and reduce the light requirements, electricity needs, and costs of CEA.Fermented wastewater is rich in key plant nutrients like nitrogen and phosphorus. However, there are secondary weak acids in fermented wastewater that can inhibit plant growth, the most prevalent weak acid being acetic acid. To be able to leverage fermented wastewater as a cheap and sustainable crop nutrient source, plants need to be bred or engineered to be more tolerant of weak acids like acetate or ideally plants would be able to actively use acetate as a chemical energy source.This research will experiment with potential yield benefits from mixotrophic growth(both light and chemical energy) and use genetic engineering and mutant screening to understand plant acetate tolerance and produce a plant line that can both tolerate and utilize acetate for energy. This would give fermented waste water a useful application as a nutrient and energy source to produce food while reducing the costs and environmental impact of CEA.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2032499104070%
2032499108030%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;
Goals / Objectives
In an effort to increase agricultural production and maximize resources devoted to food production, this research will explore increasing food production using mixotrophic growth(light and chemical energy) and utilizing new nutrient sources for food production in controlled environment agriculture(CEA).Fermented wastewater is an excellent source of nutrients such as nitrogen and phosphorus for plants, but it contains weak acids like acetate, which can inhibit plant growth. The goal of this research is to perform (1) genetic engineering and (2) a mutant screen to identify genes that are important for acetate tolerance and utilization in plants. With the ultimate goal of producing plants that can metabolize acetate as an heterotrophic energy source. This research could allow the use of fermented waste water as an alternative nutrient source for CEA and turn once inhibitory compounds into an energy source to improve crop growth and yield. An additional goal (3) is to understand the potential of mixotrophic growth by supplementing plants with sucrose and measuring any improvements in growth. These results can then be compared to any improvements in growth caused by the consumption of acetate as an energy source. Together this research works to improve resource usage and increase food production.Goal 1: Use mixotrophic growth to increase crop yield.Objective 1.1 Mixotrophic growth proof of conceptObjective 1.2 Determine the optimal amount of supplemental carbon sourceObjective 1.3 Metabolomics of plants grown with supplemented nutrients and carbon sourcesGoal 2: Forward genetic screen to improve and understand plant acetate toleranceObjective 2.1 Screening of M2 mutant Arabidopsis plants for acetate tolerance.Objective 2.2 Secondary check for false positives and establishment of strength of phenotype.Objective 2.3 Bioinformatic and functional characterization of mutantsGoal 3: Engineering approach to improving plant acetate tolerance and utilization.Objective 3.1 Over express genes in acetate utilizationObjective 3.2 Transient expression and fluorescent microscopyObjective 3.3 Characterization of phenotypes in Arabidopsis
Project Methods
Aim 1: Use mixotrophic growth to increase crop yield.Aim 1.1 Mixotrophic growth proof of concept: In a sterile environment, I will grow dwarf tomatoes in a liquid hydroponic media with the addition of sucrose (10 g/L) and controls in hydroponic solution without sucrose. Media will be changed every 15 days. I will quantify growth parameters at 10, 20, 30, 60, and 90 days after sowing (DAS). Growth parameters to be measured are germination date, plant height, plant width(radius), number of true leaves, leaves to first inflorescence, inflorescence number, flower buds, open flowers, and fruit number. At the end of the experiment, total yield, plant fresh weight, root length, fruit BRIX, fruit fresh weight and plant and fruit dry weight will be measured. Growth parameter data between controls and plants supplemented with sucrose will be compared and statistical tests performed to evaluate significant differences between treatments.Aim 1.2 Determine the optimal amount of sucrose for a plant to be supplemented with: I will evaluate the amount of sucrose that needs to be added to detect an increase in biomass and yield and whether adding more sucrose will increase plant growth and yield further or if there is an upper limit to the increase in growth. The same growth measurements and analysis as in Aim 1.1 will be performed. Aim 1.3 Metabolomics of plants grown with supplemented nutrients and carbon sources: To evaluate the incorporation of sucrose into the plant metabolome and to better understand what tissues the sucrose is incorporated into I will use stable isotope labeling. In the last 15 days of the experiment, three plants grown in sucrose will be moved into sucrose labeled with C13. This will allow quantification of sucrose incorporation into the plant metabolome. I will use the metabolomics core facilities at UCR to process my samples using LC-MS to check sucrose incorporation in leaf, fruit, and root tissues. I will also measure the sucrose concentration in the used hydroponic (plus sucrose) media using a refractometer after each media change to evaluate how much sucrose the plant is absorbing from the media. This will allow for estimation of yield (biomass (g) / sucrose (g)) and how much sucrose the plants use at different developmental time points. Expected Outcomes: I expect an increase in plant size and yield based on my preliminary findings growing tomatoes during regeneration in vitro on media supplemented with sucrose and literature that shows a rescue of plant growth in low light environments by supplemental sucrose. In Aim 1.2 I expect to find a continuum of growth improvement with a range of sucrose concentrations and an upper limit of growth. In Aim 1.3 I expect that sucrose will be incorporated very broadly into plant metabolism and that there will be higher levels of labeling in fruit as an energy sink. Pitfalls/alternative plans: If sucrose does not increase plant growth and is not strongly incorporated into plant metabolism, I will evaluate other sugars like glucose, fructose, and ribose.Aim 2: Forward genetic screen to improve and understand plant acetate toleranceAim 2.1 Screening of M2 mutant Arabidopsis plants for acetate tolerance: To improve and better understand plant acetate tolerance and utilization I will introduce genetic diversity into Arabidopsis using EMS mutagenesis and screen for increased acetate tolerance. I have produced EMS mutagenized Arabidopsis seeds, grew up 3,150 plants and let them self-pollinate to insure the emergence of any recessive mutations. I am now screening the resulting seeds of ½ MS media supplemented with 1.5 mM acetate whichinhibits the Arabidopsis growth compared to acetate free media. I am screening enough plants and seeds to get the mutation genetic coverage that should statistically yield mutants with increased acetate tolerance [26,27]. I have already screened over 10,000 seeds and have potential candidates that grew larger with longer roots on acetate supplemented media than the rest of the seeds on the screen plate. Aim 2.2 Secondary check for false positives and establishment of strength of phenotype: Candidate plants from Aim 2.1, will undergo secondary screen to identify false positives. After confirming mutant phenotypes, plants will be grown on a gradient of acetate concentrations (up to 16 mM) to examine the strength of acetate tolerance phenotypes. This will allow selection of the strongest phenotypes for further investigation.Aim 2.3 Bioinformatic and functional characterization of mutants: Causative mutations of mutant plants will be identified using genetic mapping and bulk segregant analysis, bioinformatic analysis of genes will be performed, and mutations will be complemented. Expected Outcomes: I expect to find mutants with increased acetate tolerance using this screen and then to characterize them genetically to understand the causative gene and the mechanism of increased tolerance. Pitfalls/alternative plans: Mutants with increased acetate tolerance may not be found if genes conferring acetate tolerance are involved in major biological or development mechanisms that cause seed lethality when mutated. As an alternative, Aim 3 seeks a reverse genetic approach that over-expresses genes as opposed to mutating them, is a good backup to this screen.Aim 3: Engineering approach to improving plant acetate tolerance and utilization.Aim 3.1 Over express genes in acetate utilization: There are some genes that are known to interact with acetate and process it from a weak acid into biologically active acetyl-CoA inArabidopsis (ACN1 and ACS). In fact, plants with these two genes knocked out have extreme developmental defects because the plants do not have the means to detoxify acetate that is naturally produced [28]. It has also been shown with other harmful metabolites, such as formate, that over-expressing the genes that process it in Arabidopsis increases plant tolerance of the metabolite and rescues growth inhibition [29]. I will over express these acetate utilization genes in Arabidopsis and our dwarf tomatoes using the 35S promoter to evaluate them for increased acetate tolerance and utilization. I will use Agrobacterium mediated transformation to insert these genes into tomato and Arabidopsis. I have design constructs for this purpose and some have already been made.Aim 3.2 Transient expression in tobacco and fluorescent microscopy: These constructs for over expression will include fluorescent tags (GFP and BFP) to evaluate gene expression and localization. I will use these constructs for transient expression using Agrobacterium in Nicotiana benthamian. I will then take leaf sections for confocal microscopy at the UCR microscopy core to visualize correct gene localization. ACN1 should localize to the peroxisome and ACS should localize to the chloroplast. Aim 3.3 Characterization of phenotypes: Once Arabidopsis and tomato are transformed with constructs to over-express ACN1 and ACS I will evaluate their tolerance to acetate using the same process described in Aim 2.2 and 2.3. Strength of acetate tolerance and incorporation of transformants will be determined by acetate gradient and C13 acetate metabolomics, respectively. Expected Outcomes: I expect to produce plants with increased acetate tolerance and utilization and understand the protein localization and expression levels. Increases in acetate tolerance and rates of acetate utilization will be quantified and compared to controls. Pitfalls/alternative plans: Sometimes gene expression by the 35S promoter leads to gene silencing as a plant defense response, in which case a different promoter will be used in expression constructs. If there is still low gene activity, homologs of ACN from Chlamydomonas or yeast that can already grow heterotrophically using acetate will be utilized.

Progress 01/03/22 to 01/02/25

Outputs
Target Audience:I impacted scientists in both academia and industry, as well as the general public and future scientists at UCR. I communicated my work to the public and the scientific community through the publication of our paper "A hybrid inorganic-biological artificial photosynthesis system for energy-efficient food production" in the scientific journal Nature Food in 2022. This work reached an especially broad section of the general public because it was covered by 159 news outlets and was the subject of videos by popular science YouTube channels like SciShow, which has over 7.3 million subscribers. It earned an altimetric score of 1318 and was accessed over one hundred thousand times. I also contributed to "Alternative carbon sources for the production of plant cellular agriculture: a case study on acetate," published in the scientific journal Frontiers in Plant Science in 2023. I helped publish "Electro-Agriculture: Revolutionizing Farming for a Sustainable Future" in Joule in 2024 and contributed to the development of experiments for "Optimized genome-wide CRISPR screening enables rapid engineering of growth-based phenotypes in Yarrowia lipolytica," published in Metabolic Engineering in 2024. Finally, I completed my thesis "Engineering plants for controlled environment agriculture systems by manipulating plant architecture and enabling heterotrophic growth," which was set to be published by the university and is likely to be further developed and split into two additional publications in the future. This work allowed my research to reach a broad section of society through both academic and informal channels. I gave five invited talks: one at the NCERA controlled environmental agriculture conference, one at an astrobiology seminar, one at a University of California Riverside (UCR) Center for Plant Cell Biology symposium, one at a UCR Plants3D retreat, and one at the NASA Deep Space Food Challenge award ceremony. I also gave nine talks at various conferences and events, including my PhD defense, and presented six posters at conferences such as the American Society of Plant Biology meetings. Over the course of this award, I played a major role in our lab's participation in NASA's Deep Space Food Challenge, where we proposed our technology as a means to produce food during deep space missions. Out of 300 teams from academia and industry, we made it through all three phases of the competition to the final four teams, taking our idea from concept to prototype, and all the way to a large-scale functional system operated by simulated astronauts for eight weeks at the Ohio State Food Testing Lab. We were the runners-up in the challenge, earning over $450,000 in prize money. As winners of the second phase, we attended a NASA awards ceremony in New York, where we presented our research and technology to the general public and to NASA representatives and professionals. We did the same at the final awards ceremony, where we again demonstrated our research and technology to even more professionals and space technology experts. During this award, I mentored and trained five undergraduate students and a first-year graduate student, most of whom were from underrepresented groups. Overall, I had a broad impact on a diverse range of academic and industry professionals, as well as the broader public. This would not have been possible without the support I received from this award. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Over the course of this award, I developed as a scientist and improved my technical skills, writing, data analysis, and entrepreneurship. Through my research, I learned cloning and transformation techniques for Arabidopsis and performed Confocal Microscopy to visualize my ACS-GFP overexpression lines. I conducted an EMS mutagenesis screen and optimized my screening techniques to pivot and perform another screen of the RIKEN Biosciences FOX hunting overexpression library. With the extra time and resources provided by this fellowship, I was able to conduct an RNA-seq time course under acetate treatment conditions. This opportunity allowed me to gain skills working in bioinformatics on the command line, writing scripts, and applying open-source data analysis packages to my resulting data. I was also able to greatly expand my list of targets for overexpression in Goal 3, making it much more likely that this work would have a larger impact and generate more scientific findings. I also formed collaborations through my university's Plants 3D program to develop my skills in developing a biotech product and evaluating the economic space for such a product. I was invited to give multiple talks at the Plants 3D Retreat, an event designed to help graduate students make connections between plant scientists and engineers to form collaborations and solve problems. With the help of this funding, I was able to attend multiple symposiums to learn and share my research with others, such as the American Society of Plant Biologists (ASPB) symposium in Portland, Oregon, in 2022, and the meeting in Hawaii in 2024. At the ASPB event, I presented a poster on my research, which garnered significant interest, including from industry attendees. I was able to both disseminate my work and build connections through this event. I attended the NCERA-101 symposium at UC Davis, where I gave an invited talk about our lab's research, including my publication from the previous year. I was able to both disseminate my work and build connections through this event. I also attended the 2023 Synbiobeta conference, which focused on new and developed industries in biotechnology outside of medical research. At this conference, I had a truly wonderful experience making connections and learning more about the biotechnology ecosystem that currently exists, as well as the gaps and challenges in the industry that still need to be addressed. For example, there is a huge need for increased fermenter infrastructure for producing biologics. I also developed as a teacher, leader, and manager. I had the opportunity to train and work with five undergraduates, two of whom were women in science. Both were able to contribute to this project and learned valuable technical skills in the lab, as well as how experiments are designed and conducted. This led to one of these students continuing in our lab as a technician, then leaving for a master's program in vertical agriculture, and now moving on to an internship with NASA, where they could continue to grow professionally. Research funded by this grant also helped attract new graduate students to our program, who were interested in this project or its offshoots. I had the opportunity to train these graduate students during their rotations in our lab, teaching them technical skills like qPCR and screening, while they learned about my research project. The process of acquiring the NIFA fellowship was a valuable lesson in grant writing, which I shared with other graduate students in my program by helping organize and teach a NIFA fellowship application class at UCR. This allowed me to gain experience creating a workshop and further develop my teaching and leadership skills. In addition to improving my writing through the grant-writing process, I helped write and publish a first-author manuscript on my work, "A hybrid inorganic-biological artificial photosynthesis system for energy-efficient food production," in the journal Nature Food. I also applied valuable lessons from grant writing to our lab's participation and success in NASA's Deep Space Food Challenge, in which we proposed our technology and my work as a means to produce food during deep space missions. Out of 300 teams from academia and industry, we made it through all three phases of the competition to the final four teams, taking our idea from concept to prototype, and all the way to a large-scale functional system that was operated by simulated astronauts for eight weeks to produce food at the Ohio State Food Testing Lab. We were the runners-up for the challenge and collected over $450,000 in prize money. As winners of the second phase, we attended a NASA awards ceremony in New York, where we presented our research and technology to the general public, as well as professionals and NASA representatives attending the event. We did this again at the final awards ceremony, where we won runner-up and were able to demonstrate our research and technology to many more professionals and space technology experts. I had many opportunities for training and professional development through this project and was able to enrich the scientific community while gaining experience and skills at the same time. This opportunity provided me with the time, experience, and credibility to secure additional funding for our group, allowing us to expand our work and its impact. How have the results been disseminated to communities of interest?I communicated my work to the public and the scientific community through the publication of our paper "A hybrid inorganic-biological artificial photosynthesis system for energy-efficient food production" in the scientific journal Nature Food in 2022. This work reached an especially broad section of the general public because it was covered by 159 news outlets and was the subject of videos by popular science YouTube channels like SciShow, which has over 7.3 million subscribers. It earned an altimetric score of 1318 and was accessed over one hundred thousand times. I also contributed to "Alternative carbon sources for the production of plant cellular agriculture: a case study on acetate," published in the scientific journal Frontiers in Plant Science in 2023. I helped publish "Electro-Agriculture: Revolutionizing Farming for a Sustainable Future" in Joule in 2024 and contributed to developing the experiments for "Optimized genome-wide CRISPR screening enables rapid engineering of growth-based phenotypes in Yarrowia lipolytica," published in Metabolic Engineering in 2024. Finally, I finished my thesis "Engineering plants for controlled environment agriculture systems by manipulating plant architecture and enabling heterotrophic growth," which was set to be published by the university and will likely be further developed and split into two additional publications in the future. This work allowed my research to reach an extremely broad section of society through both academic and informal channels. I gave five invited talks: one at the NCERA controlled environmental agriculture conference, one at an astrobiology seminar, one at a University of California Riverside (UCR) Center for Plant Cell Biology symposium, one at a UCR Plants3D retreat, and one at the NASA Deep Space Food Challenge award ceremony. I also gave nine talks at various conferences and events, including my PhD defense, and presented six posters at conferences such as the American Society of Plant Biology meetings. I gave annual presentations to my dissertation committee so they could mentor me and advise me on the progress of my work and methods. I also held regular lab meetings to update my peers and my mentor, Robert Jinkerson, on the progress of my research and collect feedback. I met weekly with my mentor to provide updates and receive training and guidance on the direction and results of the project. I was part of the Plants 3D program at UCR and was invited to give a talk at the Plants3D retreat on my work with acetate, as well as a collaborative project that formed with another lab at UCR as a result of our research on acetate metabolism. This opportunity allowed me to further communicate the importance and value of my research to a professional and scientific audience. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Goal 1 for mixotrophic growth was mostly completed. In vitro-grown plants with supplemental sucrose fruited in vitro, and the fruit production rate seemed to be increased with a semi-hydroponic system. I collaborated with the vertical farming company Square Roots to test acetate supplementation with plants developed in Goal 3. Supplementing plants overexpressing genes involved in acetate utilization seemed to improve plant growth under low-light conditions; however, acetate concentrations were still being optimized. Goal 1.3 was ongoing. Metabolomics was performed on some of the plants under acetate conditions, with more studies ongoing. Current results showed various changes in metabolism, such as increased succinate in root tissue, a metabolic sign of improved metabolism and growth, which paired with phenotypic observations of improved plant growth and performance under acetate. Goal 2.1 and 2.2 were accomplished; however, no obvious phenotypes were recovered from the EMS mutants, leaving us with no candidates for Goal 2.3. However, I changed my approach and began screening an overexpression Arabidopsis FOX hunting library from the RIKEN BioResource Research Center, which contained over 20,000 plant lines overexpressing individual Arabidopsis cDNAs. I finished screening this library for acetate tolerance phenotypes and recovered 21 plant lines that showed a consistent and reproducible improvement in acetate tolerance. Work on Objective 2.3 was mostly concluded. Most of the genes being overexpressed, which were likely causing the improved acetate tolerance, were identified through various methods of sequencing. A third of the identified genes were involved in plant stress responses, providing evidence that overactivating certain plant stress pathways enhanced tolerance. Another third of the identified genes had unknown molecular functions and would be the subject of further research and characterization to help understand how they played a role in acetate tolerance. Genes involved in photosynthesis, cellulose synthase, and acyl-activation were also identified. The acyl-activation gene likely had a similar effect as our engineered lines in Goal 3. The engineering approach to this problem in Goals 3.1, 3.2, and 3.3 was completed. My ACS and ACN1 overexpression lines showed statistically increased tolerance to acetate compared to wild types in many growth metrics. I expanded my targets of overexpression to create and test additional targets as well. These targets, such as AHA2, MLS, and ICL, were synthesized, cloned into their destination constructs, cloned into Agrobacterium, used to transform Arabidopsis, and homozygous lines were isolated. MLS and ICL are key genes in the glyoxylate metabolic pathway, which converts acetyl-CoA into larger carbon structures. Co-overexpressed ICL and MLS constructs did not show increased acetate tolerance but were targeted for metabolic analysis and crossed with my ACS and ACN1 overexpression lines to try to further improve acetate tolerance. There are two primary modes by which acetate can inhibit growth: acidification of the cytosol as acetic acid deprotonates upon entering a cell, or the negatively charged acetate molecule disrupting cell activities. I was able to test overactive AHA2, a hydrogen ion transporter primarily in the root. The AHA2 overactive lines did not show improved acetate tolerance, which seemed to indicate that the acetate molecule was the primary cause of stress, as opposed to cytosolic acidification. I completed, or made strong progress toward completing, many of these project goals, with some branching into slightly altered but very productive directions that led to many conclusive results and sparked new projects in my lab.

Publications

  • Type: Theses/Dissertations Status: Other Year Published: 2025 Citation: Harland-Dunaway, Marcus, 2025. "Engineering plants for controlled environment agriculture systems by manipulating plant architecture and enabling heterotrophic growth" University of California Riverside
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Crandall, Bradie S., Marcus Harland-Dunaway, Robert E. Jinkerson, and Feng Jiao. 2024. ⿿Electro-Agriculture: Revolutionizing Farming for a Sustainable Future.⿝ Joule 0 (0). https://doi.org/10.1016/j.joule.2024.09.011.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Robertson, Nicholas R., Varun Trivedi, Brian Lupish, Adithya Ramesh, Yuna Aguilar, Stephanie Carrera, Sangcheon Lee, et al. 2024. ⿿Optimized Genome-Wide CRISPR Screening Enables Rapid Engineering of Growth-Based Phenotypes in Yarrowia Lipolytica.⿝ Metabolic Engineering 86 (November): 55⿿65.


Progress 01/03/23 to 01/02/24

Outputs
Target Audience:I aim to impact scientists in both academic and industry as well as the general public and future scientists here at UCR. I have communicated my work to the public and the scientific community through publication of our work "Alternative carbon sources for the production of plant cellular agriculture: a case study on acetate" published in the scientific journal Frontiers in Plant Science. I have been able to impact undergraduates, graduate students, and professional scientists in both academic and industry through symposiums and presentations. I have given two invited talks, one to a controlled environmental agriculture conference and one to an astrobiology seminar. Our lab participated in NASA's Deep Space Food Challenge, in which we proposed our technology and my work as a means to produce food during deep space missions. We have made it through to the third and final phase of this competition that started with over 200 teams from industry and academia, after being a winner in the second phase we attend a NASA awards ceremony in New York and presented our research and technology to the general public as well as other professionals and NASA representatives attending the event. These are diverse audiences from UCR and all over the country. I have had an especially meaningful impact on undergraduates here at UCR by mentoring three undergraduates to work with me in lab. Over all I have had a broad impact on my target audiences this reporting period and will continue to do so. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During this past funding period I have developed as a scientist myself in technical skills, writing, data analysis, and entrepreneurship. Through the course of my research I have improved my cloning and transformation techniques for Arabidopsis, and conducted large scale screening techniques. With the extra time and resources provided by this fellowship I have been able to greatly expand my list of targets for overexpression making it much more likely that this work will have a larger impact and create more scientific findings. I am improving my data analysis and visualization skills while creating figures for future publications. To develop my entrepreneurial skills I have remained involved in the UCR Plants 3D program that is designed to encourage cross disciplinary collaboration and encourages participants to think about how to develop their science into products. I attended a Plants 3D retreat that had a communications workshop that trained us how to better communicate our research to broad audiences. With the help of this funding I ve been able to attend multiple symposiums to learn and also share my research with others, such as the NCERA-101 symposium at UC Davis where I gave an invited talk about our labs research including my publication form the previous year. I was able to both disseminate my work and build connections through this event. I attended the 2023 Synbiobeta, a conference focused on new and developed industries in biotechnology outside of medical research. At this conference I had a truly wonderful experience making connections and learning more about the biotechnology ecosystem that currently exists and what gaps and challenges still exist in the industry that need to be addressed, for example there is a huge need for increased fermenter infrastructure for producing biologics. I have also developed as a teacher, leader, and manager. I have had the opportunity to train and work with three undergraduates. Both have been able to contribute to this project and have learned valuable technical skills in the lab and have learned how experiments are designed and conducted. This has led to one of these students leaving our lab for a masters program in vertical agriculture, where they can continue to grow professionally. Research funded by this grant has also allowed us to attract new graduate students in our program who would be interested in this project or offshoots of this research. I have had the opportunity to train the graduate students during their rotations in our lab and taught them technical skills like qPCR and screening while they have learned about my research project. The process of acquiring the NIFA fellowship was a valuable lesson in grant writing, which I have been able to apply to our labs participation and success in NASA's Deep Space Food Challenge, in which we proposed our technology and my work as a means to produce food during deep space missions. We have made it through to the third and final phase of this competition that started with over 200 teams from industry and academia, and through this process have won over $200,000 in prize money for our research with the opportunity to get another $750,000 in this third and final phase. This allows us to continue to develop our technology system and potentially attract additional outside funders. I have had many opportunities for training and professional development through this project so far and have been able to enrich the scientific community and gain experience and skills at the same time. This opportunity has given me the time, experience, and credibility to get additional funding for our group as a whole, allowing us to expand our work and its impact. How have the results been disseminated to communities of interest?I have communicated my work to the public and the scientific community through in person events such as, NCERA-101 symposium at UC Davis where I gave an invited talk about our labs research including my publication from the previous year "A hybrid inorganic-biological artificial photosynthesis system for energy-efficient food production". I have presented posters at the Plants 3D retreat, discussed my work with other professionals at Synbiobeta 2023, and gave another invited talk for the UCR astrobiology seminar. Our lab participated in NASA's Deep Space Food Challenge, in which we proposed our technology and my work as a means to produce food during deep space missions. We have made it through to the third and final phase of this competition that started with over 200 teams from industry and academia, after being a winner in the second phase we attend a NASA awards ceremony in New York and presented our research and technology to the general public as well as other professionals and NASA representatives attending the event. I have given an annual presentation to my dissertation committee so they can mentor me and advise me on the progress of this work and my methods. I have given regular lab meetings on the progress of my research to collect feedback from my pears and my mentor Robert Jinkerson. I have met weekly with my mentor to update him and receive any training or guidance on the direction and results of the project. I was the second author on another publication this past year, "Alternative carbon sources for the production of plant cellular agriculture: a case study on acetate" published in Frontiers in Plant Science, where my work and work from our lab has been properly pear reviewed and disseminated to the broader scientific community. What do you plan to do during the next reporting period to accomplish the goals?I am working to test additional hydroponic setups and solutions to trial mixotrophic growth studies in goal 1. I have completed my initial version of goal 2 but will now pursue a modified version that is exploring a gain of function library that has resulted in at least 16 reproducible acetate resistant candidates that are under further investigation currently. For Goal 3 I have gotten homozygotic line of my ACS overexpression transformers and I am continuing to establish the phenotype and examine C13 labeled acetate uptake and incorporation in this line to complete aime 3.3. I have remade the construct to overexpress ACN1 and I am moving it through the transformation pipeline to complete goals 3.2 and 3.3 and have added additional overexpression lines through this process in parallel, which I am currently screening for homozygous lines, but the heterozygous populations showed promising resistance phenotypes already under acetate treatment conditions. Overall, all points of the project are reaching there final stages and be complete within the outlined scope within the final reporting period.

Impacts
What was accomplished under these goals? Goal 1 for mixotrophic growth is still in progress, in vitro grown plants from seed do not seem to be productive, fruit production is rare, so we have been working on optimizing conditions by working on a method to grow them in a system that is closer to hydroponics. Goal 2.1 and 2.2 were accomplished however no obvious phenotypes were recovered after screening for false positives leaving us with no candidates for Goal 2.3. So last year we changed our approach to screening a Overexpression Arabidopsis FOX hunting library from Riken BioResouce Research Center which has over 20,000 thousand plant lines overexpressing individual Arabidopsis cDNAs. I have finished screening this library for acetate tolerance phenotypes and have recovered at least 16 plant lines that show a consistent improved tolerance to acetate. I am now working on Objective 2.3 Bioinformatic and functional characterization of these overexpression lines. Goal 3 applies an engineering approach to improving acetate tolerance in plants and Goal 3.1 and 3.2 have been completed for ACS overexpression lines and progress is being made on goal 3.3. These ACS overexpression lines seem to have increased tolerance to acetate as we predicted but more testing needs to be conducted with plants that are homozygous for the overexpression cassette, which have been bred now. I have expanded my targets of overexpression to create and test as well. These targets were synthesized, cloned into their destination constructs, cloned into Agrobacterium, and used to transform Arabidopsis. I am now isolating homozygous versions of these transformants for testing, but preliminary testing of heterozygous populations also show increased acetate tolerance compared to wild type Arabidopsis. I also identified other potential gene knockouts in existing seedlines in the literature that could improve acetate tolerance and acquired these lines from the Arabidopsis Resource Center for testing. These lines did not show any significant improvement in acetate tolerance. I have made consistent progress towards completing the goals outlined here and I will continue to work to complete them in my final year of this grant.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Hann EC, Harland-Dunaway M, Garcia AJ, Meuser JE, Jinkerson RE. Alternative carbon sources for the production of plant cellular agriculture: a case study on acetate. Front Plant Sci. 2023 Oct 26;14:1104751. doi: 10.3389/fpls.2023.1104751. PMID: 37954996; PMCID: PMC10639172.


Progress 01/03/22 to 01/02/23

Outputs
Target Audience:I aim to impact scientists in both academic and industry as well as the general public and future scientists here at UCR, and in this reporting period I was very effective at doing so. I have communicated my work to the public and the scientific community through publication of our work "A hybrid inorganic-biological artificial photosynthesis system for energy-efficient food production" in the scientific journal Nature Food. This work reached an especially broad section of the general public because it was covered by 149 news outlets and was the subject of videos by very popular science youtube channels like SciShow with over 7.3 million subscribers. This allowed my work to reach an extreme broad section of society. I have been able to impact undergraduates, graduate students and professional scientists in both academic and industry through symposiums and presentations. These are diverse audiences from UCR and all over the country. I have had an especially meaningful impact on undergraduates here at UCR by bringing on two undergraduates to work with me in lab that are aspiring women in science. Over all I have had a broad impact on my target audiences this reporting period and will continue to do so. Changes/Problems:The EMS screen I performed in arabidopsis did not yield any candidate mutants after screening for false positives. Instead of giving up on this approach I have acquired an overactivation library(the Fox hunting system) from Riken BioResource center in Japan that has about 21000 overexpression lines of individual Arabidopsis cDNAs. I worked with USDA APHIS to get a permit to import these seeds, and now have the library in hand to start screening. I had trouble cloning and expressing ACN1 from Goal 3, so I acquired new constructs and synthesized a new version for expression. This is now being moved through the transformation pipeline and I should be able to report on its phenotype during the next reporting period. What opportunities for training and professional development has the project provided?During this past funding period I have developed as a scientist myself in technical skills, writing, data analysis, and entrepreneurship. Through the course of my research so far I have learned cloning and transformation techniques for Arabidopsis and have been doing Microscopy to visualize my ACS overexpression lines that have been linked to a GFP tag. I have conducted an EMS mutagenisis screen and have optimized my screening techniques. With the extra time and resources provided by this fellowship I have been able to conduct an RNAseq time course under acetate treatment conditions. This opportunity has allowed me to gain skills working in bioinformatics on the command line writing scripts and applying open source data analysis packages to my resulting data. I have also been able to form collaborations through my university's Plants 3D program to develop my skills in developing a biotech product and evaluating the economic space for such a product. I was even invited to give a talk at the Plants 3D Retreat, an event which helps graduate students make connections between plant scientists and engineers to form collaborations and solve problems. With the help of this funding I ve been able to attend multiple symposiums to learn and also share my research with others, such as the American Society of Plant Biologists (ASPB) symposium in Portland Oregon. At the ASPB event I was able to present a poster on my research which many people were very interested in, including attendees from industry. I was able to both disseminate my work and build connections through this event. I have also developed as a teacher, leader, and manager. I have had the opportunity to train and work with two undergraduate women in science. Both have been able to contribute to this project and have learned valuable technical skills in the lab and have learned how experiments are designed and conducted. This has led to one of these students continuing in our lab as a technician, where they can continue to grow professionally. Research funded by this grant has also allowed us to attract new graduate students in our program who would be interested in this project or offshoots of this research. I have had the opportunity to train the graduate students during their rotations in our lab and taught them technical skills and provided an opportunity to learn about my research project. The process of acquiring the NIFA fellowship was a valuable lesson in grant writing, which I have been able to share with other graduate students in my program by helping to organize and teach a NIFA fellowship application class at UCR. This allowed me to get experience creating a workshop, and gain more teaching and leadership experience. In addition to improving my writing through the grant writing process I helped to write and publish a first author manuscript on my work "A hybrid inorganic-biological artificial photosynthesis system for energy-efficient food production" in the journal Nature Food. I have had many opportunities for training and professional development through this project so far and have been able to enrich the scientific community and gain experience and skills at the same time. How have the results been disseminated to communities of interest?I have communicated my work to the public and the scientific community through publication of our work "A hybrid inorganic-biological artificial photosynthesis system for energy-efficient food production" in the scientific journal Nature Food. Our paper was covered by 149 news outlets and was the subject of videos by very popular science youtube channels like SciShow with over 7.3 million subscribers. Our work has been broadly covered by sources that communicated it to the general population in an understandable manner. I have also communicated my research to the scientific community through poster presentations at ASPB and at UCR for the Delfino foundation. I have also presented oral presentations such as one I made during a Plants3D program retreat at Lake arrowhead. I have given an annual presentation to my dissertation committee so they can mentor me and advise me on the progress of this work and my methods. I have given regular lab meetings on the progress of my research to collect feedback from my pears and my mentor Robert Jinkerson I have met weekly with my mentor to update him and receive any training or guidance on the direction and results of the project. I am a part of the Plants 3D program at UCR and I was invited to give a talk at the Plants3D retreat on my work with acetate and a collaborative project that has formed with another lab at UCR as a product of our research on acetate metabolism. This allowed me to further communicate the importance and value of my research to a professional and scientific audience. With the help of this funding I ve been able to attend multiple symposiums to learn and also share my research with others, such as the American Society of Plant Biologists (ASPB) symposium in Portland Oregon. At the ASPB event I was able to present a poster on my research which many people were very interested in, including attendees from both academia and industry. I was able to both disseminate my work and build connections through this event. What do you plan to do during the next reporting period to accomplish the goals?I am working to test additional hydroponic setups and solutions to trial mixotrophic growth studies in goal 1. I have completed my initial version of goal 2 but will now pursue a modified version that is exploring a gain of function library. For Goal 3 I will finish getting a homozygotic line of my ACS overexpression transformat and establishing the phenotype and examine C13 labeled acetate uptake and incorporation in this line to complete aime 3.3. I have remade the construct to overexpress ACN1 and I am moving it through the transformation pipeline to complete goals 3.2 and 3.3. I have also selected some additional overexpression lines to create and test which have been synthesized and are being cloned and moved through the transformation pipeline as well.

Impacts
What was accomplished under these goals? Goal 1 for mixotrophic growth is still in progress, in vitro grown plants from seed do not seem to be productive, fruit production is rare, so we have been working on optimizing conditions by working on a method to grow them in a system that is closer to hydroponics. Goal 2.1 and 2.2 were accomplished however no obvious phenotypes were recovered after screening for false positives leaving us with no candidates for Goal 2.3. However, it could be that because EMS mutagenesis is more likely to cause loss of function mutations it is a less effective screening library for our purpose of increasing acetate tolerance. Following this reasoning, for Goal 2 I have acquired the Overexpression Arabidopsis FOX hunting library from Riken BioResouce Research Center which has thousands of plant lines overexpressing individual Arabidopsis cDNAs and I have begun screening this library for more acetate tolerance phenotypes. Applying an engineering approach to this problem in Goal 3.1 and 3.2 have been completed for ACS overexpression lines and progress is being made on goal 3.3. These ACS overexpression lines seem to have increased tolerance to acetate as we predicted but more testing needs to be conducted with plants that are homozygous for the overexpression cassette. However there were complications trying to overexpress ACN1 so it will be transformed with an alternative construct design and additionally I have expanded my targets of overexpression to create and test, I have had these targets synthesized and they are now being moved through the cloning and transformation pipeline. I have also identified other potential genetic manipulations in existing seedlines in the literature that could improve acetate tolerance and I have moved forward to acquire the available lines for testing. Significant progress has been made towards these goals and I will continue to work to complete these goals and the work that has expanded around them.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Hann, E.C., Overa, S., Harland-Dunaway, M. et al. A hybrid inorganicbiological artificial photosynthesis system for energy-efficient food production. Nat Food 3, 461471 (2022). https://doi.org/10.1038/s43016-022-00530-x