UNDERSTANDING PLANT NATURAL PRODUCT BIOSYNTHESIS IN BLUEBERRY THROUGH CORE GENE DISCOVERY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028055
• Grant No.: 2022-67013-36416
• Cumulative Award Amt.: $281,173.00
• Proposal No.: 2021-07820
• Project Start Date: Jan 15, 2022
• Project End Date: Jan 14, 2025
• Grant Year: 2022
• Program Code: [A1103]- Foundational Knowledge of Plant Products

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
Biological Sciences

Non Technical Summary
Food crops provide the essential micronutrients needed in the human diet through production of minerals and vitamins. Additionally, plants produce thousands of specialized metabolites, many of which have human health benefits. The Ericaceae family contains several economically important fruit crops that produce diverse natural products, including blueberry and cranberry. Cultivated blueberry has seen an annual increase in market size since 2000, with rising popularity due to the overall sweet fruit flavor and consumer perceived levels of known health-promoting compounds. Blueberries also produce iridoid compounds, which are potent natural products with human health benefits. Despite these known properties, little work has been done to fully understand the genetic basis of iridoid biosynthesis in cultivated blueberry. This New Investigator Seed Grant aims to obtain data to identify core iridoid biosynthetic genes in blueberry. A multi-faceted approach incorporating transcriptomic, genomic and metabolomic data will be used to elucidate genes involved in the biosynthesis of the iridoid glycoside monotropein in blueberry. The objectives of the proposal are: 1) Identify iridoid biosynthetic pathway genes in blueberry using transcriptomic, orthology, co-expression and genome sequencing analysis, and 2) Validate the computationally predicted gene targets through functional enzyme characterization. This work leverages the within-species genotypic and phenotypic diversity in blueberry iridoid production to understand the genetic basis of natural product biosynthesis in this economically important crop. Outcomes from this proposal will serve as a platform to more fully understand iridoid production in blueberry and can translate into the development of new blueberry varieties with enhanced human health benefits.

Animal Health Component

10%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1120	1080	50%
206	1120	1000	50%

Knowledge Area
206 - Basic Plant Biology; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1120 - Blueberry;

Field Of Science
1000 - Biochemistry and biophysics; 1080 - Genetics;

Keywords

natural products

blueberry

gene discovery

iridoids

seed grant

Goals / Objectives
The research in this proposal addresses the genetic basis of the synthesis of plant-derived, high-value chemicals in blueberries. As part of the balanced human diet blueberries can provide essential micronutrients, vitamins, and thousands of specialized metabolites with additional health benefits. The Ericaceae plant family contains several economically important fruit crops including blueberry and cranberry. Cultivated blueberry (Vacciniumspecies) has seen an annual increase in U.S. market size since 2000. The popularity of this crop is based on their overall sweet flavor of the fruit and the consumer perceived levels of known health-promoting compounds, including vitamin C, folate, phenolic and flavonoid compounds.Blueberries also produce iridoid compounds, which are potent natural products with human health benefits. Several iridoid compounds have been identified in a subset of species within the Ericaceae, with significant inter-species diversity observed in production of the iridoid glycoside monotropein in blueberry.Despite the relevant human health properties, little work has been done to fully understand the iridoid biosynthetic pathway in cultivated blueberry.This New Investigator Seed Grant aims to address this knowledge gap by obtaining data to identify core iridoid biosynthetic genes in blueberry.Themajor hypothesisof this work is that the genes involved in iridoid biosynthesis in blueberry can be identified by comparative genomics and orthology analysis using specific blueberry varieties known to have presence/absence of this class of secondary metabolites.A multi-faceted approach incorporating transcriptomic, genomic and metabolomic data will be used to elucidate genes involved in the biosynthesis of the iridoid glycoside monotropein in cultivated blueberry. This work leverages the within-species genotypic and phenotypic diversity in blueberry iridoid production to understand the genetic basis of natural product biosynthesis in this economically important crop.The two objectives of the proposed project are:1)Identify iridoid biosynthetic pathway genes in blueberryusing transcriptomic, orthology, co-expression and genome sequence analysis; and2)Validate the computationally predicted gene targets through functional enzyme characterization.

Project Methods
Objective 1:Identify iridoid biosynthetic pathway genes in blueberryHypothesis: The combination of analyses using transcriptomics, orthology, co-expression and genome sequence analysis will reveal the candidate iridoid biosynthesis genes.Objective 1, Task 1.A new cultivar and tissue panel will be generated for select cultivars to expand the genetic and phenotypic diversity needed for identification of monotropein biosynthetic genes. Both Ornablue and Concord cultivars have been previously analyzed for monotropein content in several tissues. We already havede novotranscriptome assemblies completed for tissues from Ornablue (+) and Concord (-) which can be used for candidate gene identification using orthology analysis to previously characterized iridoid biosynthetic genes inC. roseus. Three additional cultivars 1) Summit, a previously identified monotropein-positive cultivar that is a southern highbush ecotype (Leisner et al., 2017) 2) Cara's choice (monotropein-negative), a half-sibling of Summit (Ehlenfeldt & Stretch, 2005) and northern highbush ecotype, and 3) Tifblue, a rabbiteye cultivar (V. virgatum)have been selected for analysis to expand the panel diversity. Tifblue is a late season blueberry variety that is one of the most important cultivars in rabbiteye breeding (Hancock et al., 2008).Rabbiteye blueberries are native to the southeastern U.S. and a major ecotype grown in Alabama and southern Georgia (Coneva, 2013). In this new expanded panel, root tissue will be analyzed for all cultivars as other plant species has shown significant production of iridoids in plant roots (e.g., Kellner et al., 2015). As the origin and/or transport of monotropein production in blueberry are unknown, analysis of root tissue is invaluable. This panel will also include young leaves sprayed with methyl jasmonate (MeJa).Previous work has demonstrated that application of MeJa can increase the level of monoterpenes in cranberry (Rodriguez-Saona et al., 2013),C. roseus(Liscombe et al., 2011), and other species (Wang et al., 2010; Nagatoshi et al., 2011; Góngora-Castillo et al., 2012; Semiz et al., 2012). It is hypothesized that increasing monotropein production will increase expression and/or abundance of genes related to iridoid biosynthesis and be a valuable asset in gene discovery and co-expression analysis.All samples for the blueberry tissue diversity panel will be collected from verified cultivars in conjunction with theNorth Alabama Horticulture Research Center and Chilton Research & Extension Center as well as with support from Dr. Sushan Ru.Monotropein content of ground tissues will be analyzed and quantified by targeted LC-MS using an authentic standard as described in Leisner et al. (2017) (also see Letter of Collaboration from Dr. Sarah O'Connor). For transcriptomic analysis, RNA will be isolated and cDNA libraries will be constructed and sequenced with the Illumina HiSeq4000 sequencer. In addition to short-read sequencing,transcriptome assemblies for all cultivars will be reconstructed using isoform sequencing (Iso-Seq) from Pacific Biosciences (PacBio). Short-read Illumina data generated from the HiSeq4000 will be used to error correct the reconstructed transcriptomes and generate gene expression data.From the Iso-Seq analysis a high-quality, full-length transcripts will be generated with no assembly required. Gene expression analysis will be completed by mapping each RNA-Seq library to the transcriptome for each cultivar followed by determination of transcript abundance values.Objective 1, Task 2.Enzymes in threekey steps in the early iridoid biochemical pathway will be selected for targeted orthology analysis - GES, G8H and GOR. These genes represent the first committed steps in iridoid biosynthesis (Miettinen et al., 2014) and should share sufficient homology withC. roseusto utilize comparative genomic and orthology analysis. We will also use the new data to validate our previous work with ISY.Orthology analysis will be completed and potential orthologs will be analyzed for presence/absence variants and transcript abundance by mapping the transcript sequences to theV. corymbosumgenome (cv. Draper, Colle et al., 2019) and to the transcriptome assembly for each cultivar.Co-expression analyses, including hierarchical clustering, principal component analyses, mutual rank, and self-organizing maps will be used to identify the candidate genes involved in the early iridoid biosynthetic pathway (Obayashi & Kinoshita, 2009; Saeed et al., 2003; Wehrens & Buydens, 2007).Anticipated results and interpretations:In objective 1 transcriptomic data and bioinformatic analyses will be used to identify three genes involved in the early iridoid pathway in blueberry (GES, G8H, GOR). Functional annotation, co-expression analyses, and physical clustering of genes within the genome will be used to identify key enzymes involved in biosynthesis of monotropein produced in blueberry. These core genes will be further characterized in objective 2. Objective 1 will produce a wealth of transcriptomic data that can be used in future work to search for the downstream genes involved in monotropein biosynthesis, furthering our understanding of iridoid biosynthesis in blueberry.Objective 2: Validate the computationally predicted gene targets through functional enzyme characterizationHypothesis:The functionality of the gene products can be confirmed by biochemical analysis.Objective 2, Task 1.In order to validate the computationally predicted gene targets, cloning of genes encoding three key enzymes (GES, G8H, GOR) will be completed for two blueberry genotypes (Table 1), with the long-term goal of confirming enzyme sequences for all cultivars (outside the scope of this grant). The entire coding sequence will be amplified and cloned into pOPINF expression vector using In-Fusion cloning (Takara Bio). Plasmids containing the cloned gene will be transformed into competentE. colior yeast expression strain cells to produce protein in quantities suitable for enzymatic analysis. After lysing the cells by sonication, the soluble portion will be loaded onto Ni-NTA agarose resin (Qiagen), and the recombinant His-tagged protein eluted. The purified recombinant protein will then be used in Task 2.Objective 2, Task 2.To validate enzyme function, end-point enzyme assays for the enzymes of interest will be done according to Lichman et al., 2020. This is key to confirming the cloned gene sequence produces a protein that has the assigned function in the iridoid biosynthetic pathway. To obtain anin vitroprofile of reaction products for the enzyme of interest GC-MS will be performed(Yang et al., 2009; Geu-Flores et al., 2012). The early biosynthetic intermediates, such as geranyl diphosphate, geraniol, and 8-hydroxygeraniol required as a substrate for these enzymes are commercially available. Completion of this objective will be done in collaboration with Dr. Sarah O'Connor, Director at the Max Planck Institute for Chemical.Anticipated results and interpretations:Co-expression analyses and orthology analysis in Objective 1 will be used to determine candidate genes sequences of interest in Objective 2 for cloning and functional enzyme characterization. Using a heterologous expression system and GC-MS/LC-MS to determine iridoid profiles, protein products for candidate genes of interest will be used to confirm their role in the monotropein biosynthetic pathway.

Progress 01/15/22 to 01/14/25

Outputs
Target Audience:There are several target audiences that are the focus of effort for the duration of this USDA NIFA Project. They include: Early Career Researchers and Students: Undergraduate students, graduate students, and a postdoctoral researcher have been served by this project through direct and indirect involvement in research projects, data analysis and presentation of their research findings. The postdoctoral researcher worked with one undergraduate researcher on this project during the spring semester of 2023. This involved regular meetings regarding the project progress and help with troubleshooting. Breeders: Through collaboration with the small fruit breeder at Auburn University in the Department of Horticulture we work with them to directly translate the research findings in a way that will facilitate future breeding efforts focused on natural product biosynthesis in blueberry. Broader scientific community: The findings from this work will be disseminated through conference presentations and open-access peer-reviewed publications. Additionally, data will be deposited in public repositories (NCBI and Github). This will allow the larger scientific community access to our research findings and increase understanding and awareness of natural product biosynthesis in blueberry.Students will also be directly involved in outreach opportunities that will serve the local community. For example, the graduate student involved in the project and PI Leisner participated in an annual outreach event in the College of Sciences and Mathematics entitled "EmpowHER 2023". This is a mini-conference aimed at encouraging and building confidence of 8-10th grade girls planning to pursue STEM majors in college. During this event we interacted with students and explained how plants are chemical factories and the ways in which we benefit from plant specialized metabolites. Changes/Problems:During the Year 2 project period PI Leisner left Auburn University and began a new position at Virginia Tech in The School of Plant and Environmental Sciences (August 2023). During this time there was a natural slowdown of wet lab work, and a subsequent ramping up period while the lab was set-up at Virginia Tech. The wet lab is now functional at Virginia Tech. PI Leisner was able to submit a large amount of data for analysis (metabolite and sequencing) to ensure significant progress on the project while the wet lab was unavailable. Additionally, the graduate student who took over the project after the departure of the postdoctoral researcher moved to Virginia Tech as well, ensuring the continuation of the proposal work. We have also found GES to be somewhat recalcitrant to cloning and had the gene synthesized. This also did not produce sufficient protein for functional characterization. We will trouble shoot alternative cloning approaches and work on cloning other genes in the pathway. We are optimistic the IsoSeq data will provide a more full-length sequence for GES, as well as other targets for cloning. The mutual rank analysis which accompanied the ISY cloning paper inPlant Directalso identified some promising targets for cloning and functional characterization. What opportunities for training and professional development has the project provided?Dr. Leisner has provided teaching and mentoring to the students and postdoc involved in the project. Dr. Leisner has provided them with the skills and learning environment to further their own curiosity and research endeavors in natural product biosynthesis in blueberry. Dr. Leisner had a teaching release in Spring 2023 and has not taught any courses during the current time frame, but plants on teaching a laboratory course focused on plant physiology in 2024 which provides further training for students. Additionally, Dr. Leisner meets weekly with the researchers on the project (currently a graduate student) to discuss project goals, recent progress, project hurdles and next steps. This gives the graduate student on the project an opportunity to discuss their own scientific questions and practice their scientific communication skills. Prior to June 2023 PI Leisner also met weekly with the postdoctoral researcher on the project as well. Additionally, the postdoctoral researcher met weekly with the undergraduate students on the project to assess their progress and aid in troubleshooting (during Spring 2023). How have the results been disseminated to communities of interest?All data produced from this proposal will be publicly available through data servers after publication, data repositories (i.e., GitHub), and federal data repositories (NCBI and SRA). Funds from this project will also be used to provide open access to all future publications to make them freely available to the public. The research conducted under this project (completed by the PI, graduate student, undergraduates, and postdoctoral researcher) was presented at one meeting and three invited seminars during the second year of the grant. Leisner CP (2023) Invited Talk, "Medicinal Genomics: Exploring natural products in blueberry for human health benefits". American Society of Plant Biology Annual Meeting, Savannah, Georgia. Leisner CP (2023) Invited Seminar, "Understanding natural product biosynthesis in blueberries: a focus on iridoids". Harrison School of Pharmacy, Auburn University. Leisner CP (2023) Invited Seminar, "Climate change impacts on plant physiology". Green Group, Department of Biological Sciences, Florida State University.? Leisner CP (2023) Invited Seminar, "Climate change impacts on plant physiology". Ball Horticultural Company.? What do you plan to do during the next reporting period to accomplish the goals?We are requesting a no-cost extension to continue the work proposed in the grant. During this time, we will continue to screen cultivars of blueberry for the presence of monotropein (Objective 1). Additionally, we will analyze the short-read RNA-Sequencing and long-read Iso-seq data we have just received for gene discovery (Objective 1). Finally, we will continue work to clone and functionally characterize key genes in the monotropein biosynthetic pathway in blueberry (Objective 2).Additionally, the data from this work will be presented at the American Society of Plant Biology meeting in summer 2024 and the Botanical Society of America meeting in summer 2024. We will also work on generating a new proposal for submission to the USDA AFRI program to continue the work on natural product biosynthesis in blueberry leveraging results from this grant.

Impacts
What was accomplished under these goals? Several experiments have been completed in Year 2. First, we have completed cloning and functional characterization of the ISY gene in blueberry. Subsequently, we performed mutual rank and other co-expression analysis to determine the role of transcript expression on the production of monotropein blueberry. This work has been published as a peer-reviewed manuscript inPlant Direct. We are now working on cloning another gene in the pathway, GES, which is a key step differentiation the production of monoterpenes and iridoid compounds (Objective 2). This work was initiated by an undergraduate student in the laboratory under the supervision of the postdoctoral researcher and is now being continued by the postdoctoral researcher in the laboratory. In June 2023 the postdoctoral researcher left the laboratory for a new position and this work has been taken over by a graduate student. Additionally, the postdoctoral has screened over 20 additional blueberry fruit and leaf samples for the presence/absence of monotropein (Objective 1). These include the new cultivars we proposed to screen in the grant proposal. Interestingly, the rabbiteye ecotype of blueberry seems to produce monotropein in most of the cultivars we survey, which may be a potential new strategy for breeding. Additionally, a wildVacciniumaccession (V. darrowii) has been shown to produce iridoids. This is of note since there is now a publicly available genome forV. darrowii. This will help immensely in the gene discovery process. The postdoctoral researcher with help from the graduate student on the project also submitted samples for short-readand long-read (Iso-seq) RNA-Sequencing. This will aid in gene discovery as it will not require transcriptome assembly and hopefully derive full-length transcript information for cultivars we do not have a genome available for (Objective 1). Currently, the graduate student is analyzing the RNA-Sequencing data for differential expression and preparing the Iso-seq data for orthology analysis to identify potentially new or improved transcript sequences for genes in the monotropein biosynthetic pathway. Additionally, we have screened two cultivars for monotropein content in roots. We had not previously screened root tissues. Preliminary data indicates roots produce significantly higher amounts of monotropein than even floral bud tissue, which is similar in finding to work done inCatharanthus. Additionally, we screened plants grown under ambient and elevated temperature for monotropein and found elevated temperature decreases monotropein content. The effects of temperature on monotropein is tangential to the grant objectives but still increases our understanding of biosynthesis of metabolites in blueberry with human health benefits. We have also worked with the USDA germplasm repository in Corvalis, Oregon to obtain young leaf and mature fruit from a suite of wildVacciniumgermplasm to screen for the presence of monotropein. We will then use these tissues along with the fruits from cultivated blueberry to run untargeted LC-MS to determine if monotropein affects the flavor profile of blueberry. While outside the scope of the current proposal, it is generating data for future grant proposals with AFRI. Finally, we are also using the preliminary data from this grant to develop a collaboration with a researcher at Virginia Tech (Dr. Dennis Cladis) to look at the bioaccessibility and bioavilability of monotropein in blueberry fruit. An initial proposal based on this collaboration was submitted to the USDA AFRI program A1343 Food and Human Health entitled "Blueberries and bone health: Characterizing the metabolome and gut microbiome of the novel phytochemical monotropein" (PI Dennis Cladis, Co-PI Leisner).

Publications

• Type: Journal Articles Status: Other Year Published: 2024 Citation: Lawas LMF, Rossi G, D, Tian D, Spiers J, Salazar-Gutierrez M, Leisner CP (In prep) Temperature impacts on bud break in blueberry.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Lawas LMF, Kamileen MO, Buell CR, OConnor SE, Leisner CP (2023) Transcriptome-based identification and functional characterization of iridoid synthase involved in monotropein biosynthesis in blueberry. Plant Direct.7:e512.

Progress 01/15/22 to 11/06/23

Outputs
Target Audience:There are several target audiences that are the focus of effort for the duration of this USDA NIFA Project. They include: Early Career Researchers and Students: Undergraduate students, graduate students, and a postdoctoral researcher have been served by this project through direct and indirect involvement in research projects, data analysis and presentation of their research findings. The postdoctoral researcher worked with one undergraduate researcher on this project during the spring semester of 2023. This involved regular meetings regarding the project progress and help with troubleshooting. Breeders: Through collaboration with the small fruit breeder at Auburn University in the Department of Horticulture we work with them to directly translate the research findings in a way that will facilitate future breeding efforts focused on natural product biosynthesis in blueberry. Broader scientific community: The findings from this work will be disseminated through conference presentations and open-access peer-reviewed publications. Additionally, data will be deposited in public repositories (NCBI and Github). This will allow the larger scientific community access to our research findings and increase understanding and awareness of natural product biosynthesis in blueberry.Students will also be directly involved in outreach opportunities that will serve the local community. For example, the graduate student involved in the project and PI Leisner participated in an annual outreach event in the College of Sciences and Mathematics entitled "EmpowHER 2023". This is a mini-conference aimed at encouraging and building confidence of 8-10th grade girls planning to pursue STEM majors in college. During this event we interacted with students and explained how plants are chemical factories and the ways in which we benefit from plant specialized metabolites. Changes/Problems:During the Year 2 project period PI Leisner left Auburn University and began a new position at Virginia Tech in The School of Plant and Environmental Sciences (August 2023). During this time there was a natural slowdown of wet lab work, and a subsequent ramping up period while the lab was set-up at Virginia Tech. The wet lab is now functional at Virginia Tech. PI Leisner was able to submit a large amount of data for analysis (metabolite and sequencing) to ensure significant progress on the project while the wet lab was unavailable. Additionally, the graduate student who took over the project after the departure of the postdoctoral researcher moved to Virginia Tech as well, ensuring the continuation of the proposal work. We have also found GES to be somewhat recalcitrant to cloning and had the gene synthesized. This also did not produce sufficient protein for functional characterization. We will trouble shoot alternative cloning approaches and work on cloning other genes in the pathway. We are optimistic the IsoSeq data will provide a more full-length sequence for GES, as well as other targets for cloning. The mutual rank analysis which accompanied the ISY cloning paper inPlant Directalso identified some promising targets for cloning and functional characterization. What opportunities for training and professional development has the project provided?Dr. Leisner has provided teaching and mentoring to the students and postdoc involved in the project. Dr. Leisner has provided them with the skills and learning environment to further their own curiosity and research endeavors in natural product biosynthesis in blueberry. Dr. Leisner had a teaching release in Spring 2023 and has not taught any courses during the current time frame, but plants on teaching a laboratory course focused on plant physiology in 2024 which provides further training for students. Additionally, Dr. Leisner meets weekly with the researchers on the project (currently a graduate student) to discuss project goals, recent progress, project hurdles and next steps. This gives the graduate student on the project an opportunity to discuss their own scientific questions and practice their scientific communication skills. Prior to June 2023 PI Leisner also met weekly with the postdoctoral researcher on the project as well. Additionally, the postdoctoral researcher met weekly with the undergraduate students on the project to assess their progress and aid in troubleshooting (during Spring 2023). How have the results been disseminated to communities of interest?All data produced from this proposal will be publicly available through data servers after publication, data repositories (i.e., GitHub), and federal data repositories (NCBI and SRA). Funds from this project will also be used to provide open access to all future publications to make them freely available to the public. The research conducted under this project (completed by the PI, graduate student, undergraduates, and postdoctoral researcher) was presented at one meeting and three invited seminars during the second year of the grant. Leisner CP (2023) Invited Talk, "Medicinal Genomics: Exploring natural products in blueberry for human health benefits". American Society of Plant Biology Annual Meeting, Savannah, Georgia. Leisner CP (2023) Invited Seminar, "Understanding natural product biosynthesis in blueberries: a focus on iridoids". Harrison School of Pharmacy, Auburn University. Leisner CP (2023) Invited Seminar, "Climate change impacts on plant physiology". Green Group, Department of Biological Sciences, Florida State University.? Leisner CP (2023) Invited Seminar, "Climate change impacts on plant physiology". Ball Horticultural Company.? What do you plan to do during the next reporting period to accomplish the goals?We are requesting a no-cost extension to continue the work proposed in the grant. During this time, we will continue to screen cultivars of blueberry for the presence of monotropein (Objective 1). Additionally, we will analyze the short-read RNA-Sequencing and long-read Iso-seq data we have just received for gene discovery (Objective 1). Finally, we will continue work to clone and functionally characterize key genes in the monotropein biosynthetic pathway in blueberry (Objective 2).Additionally, the data from this work will be presented at the American Society of Plant Biology meeting in summer 2024 and the Botanical Society of America meeting in summer 2024. We will also work on generating a new proposal for submission to the USDA AFRI program to continue the work on natural product biosynthesis in blueberry leveraging results from this grant.

Impacts
What was accomplished under these goals? Several experiments have been completed in Year 2. First, we have completed cloning and functional characterization of the ISY gene in blueberry. Subsequently, we performed mutual rank and other co-expression analysis to determine the role of transcript expression on the production of monotropein blueberry. This work has been published as a peer-reviewed manuscript inPlant Direct. We are now working on cloning another gene in the pathway, GES, which is a key step differentiation the production of monoterpenes and iridoid compounds (Objective 2). This work was initiated by an undergraduate student in the laboratory under the supervision of the postdoctoral researcher and is now being continued by the postdoctoral researcher in the laboratory. In June 2023 the postdoctoral researcher left the laboratory for a new position and this work has been taken over by a graduate student. Additionally, the postdoctoral has screened over 20 additional blueberry fruit and leaf samples for the presence/absence of monotropein (Objective 1). These include the new cultivars we proposed to screen in the grant proposal. Interestingly, the rabbiteye ecotype of blueberry seems to produce monotropein in most of the cultivars we survey, which may be a potential new strategy for breeding. Additionally, a wildVacciniumaccession (V. darrowii) has been shown to produce iridoids. This is of note since there is now a publicly available genome forV. darrowii. This will help immensely in the gene discovery process. The postdoctoral researcher with help from the graduate student on the project also submitted samples for short-readand long-read (Iso-seq) RNA-Sequencing. This will aid in gene discovery as it will not require transcriptome assembly and hopefully derive full-length transcript information for cultivars we do not have a genome available for (Objective 1). Currently, the graduate student is analyzing the RNA-Sequencing data for differential expression and preparing the Iso-seq data for orthology analysis to identify potentially new or improved transcript sequences for genes in the monotropein biosynthetic pathway. Additionally, we have screened two cultivars for monotropein content in roots. We had not previously screened root tissues. Preliminary data indicates roots produce significantly higher amounts of monotropein than even floral bud tissue, which is similar in finding to work done inCatharanthus. Additionally, we screened plants grown under ambient and elevated temperature for monotropein and found elevated temperature decreases monotropein content. The effects of temperature on monotropein is tangential to the grant objectives but still increases our understanding of biosynthesis of metabolites in blueberry with human health benefits. We have also worked with the USDA germplasm repository in Corvalis, Oregon to obtain young leaf and mature fruit from a suite of wildVacciniumgermplasm to screen for the presence of monotropein. We will then use these tissues along with the fruits from cultivated blueberry to run untargeted LC-MS to determine if monotropein affects the flavor profile of blueberry. While outside the scope of the current proposal, it is generating data for future grant proposals with AFRI. Finally, we are also using the preliminary data from this grant to develop a collaboration with a researcher at Virginia Tech (Dr. Dennis Cladis) to look at the bioaccessibility and bioavilability of monotropein in blueberry fruit. An initial proposal based on this collaboration was submitted to the USDA AFRI program A1343 Food and Human Health entitled "Blueberries and bone health: Characterizing the metabolome and gut microbiome of the novel phytochemical monotropein" (PI Dennis Cladis, Co-PI Leisner).

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Lawas LMF, Kamileen MO, Buell CR, OConnor SE, Leisner CP (2023) Transcriptome-based identification and functional characterization of iridoid synthase involved in monotropein biosynthesis in blueberry. Plant Direct.7:e512.
• Type: Journal Articles Status: Other Year Published: 2024 Citation: Lawas LMF, Rossi G, D, Tian D, Spiers J, Salazar-Gutierrez M, Leisner CP (In prep) Temperature impacts on bud break in blueberry.

Progress 01/15/22 to 01/14/23

Outputs
Target Audience:There are several target audiences that are the focus of effort for the duration of this USDA NIFA Project. They include: Early Career Researchers: This includes early career researchers through both teaching and research mentoring. Undergraduate students and a postdoctoral researcher have been served by this project through direct and indirect involvement in research projects, data analysis, and presentation of their research findings. Undergraduate students are also served by this project through formal teaching at Auburn University through the "Advanced Plant Physiology" course taught annually. Students will also be directly involved in outreach opportunities that will serve the local community. For example, the postdoctoral researcher involved in the project participated in an annual outreach event in the College of Sciences and Mathematics entitled "GUTS: Getting Under The Surface". During this event, they interacted with elementary school students and exposed them to plant science by interacting with fruits and vegetables through microscopy and DNA extractions. Breeders: Through collaboration with the small fruit breeder at Auburn University in the Department of Horticulture we work with them to directly translate the research findings in a way that will facilitate future breeding efforts focused on natural product biosynthesis in blueberry. Broader scientific community: The findings from this work will be disseminated through conference presentations and open-access peer-reviewed publications. Additionally, data will be deposited in public repositories (NCBI and Github). This will allow the larger scientific community access to our research findings and increase understanding and awareness of natural product biosynthesis in blueberries. Changes/Problems:We have found GES to be somewhat recalcitrant to cloning and had the gene synthesized. This also did not produce sufficient protein for functional characterization. We will troubleshoot alternative cloning approaches and work on cloning other genes in the pathway. Additionally, the postdoctoral researcher on the project will be leaving the lab to pursue a new position in June 2023. A graduate student will take over the remaining aspects of the project. What opportunities for training and professional development has the project provided?Dr. Leisner has provided teaching and mentoring to the students and postdoc involved in the project. Dr. Leisner has provided them with the skills and learning environment to further their own curiosity and research endeavors in natural product biosynthesis in blueberry. Dr. Leisner has also taught a course focused on plant physiology which provides further training for students. Additionally, Dr. Leisner meets weekly with the postdoctoral researcher on the project to discuss project goals, recent progress, project hurdles and next steps. This gives the postdoctoral researcher on the project an opportunity to discuss their own scientific questions and practice their scientific communication skills.The postdoctoral researcher also meets weekly with the undergraduate students on the project to assess their progress and aid in troubleshooting. How have the results been disseminated to communities of interest?All data produced from this proposal will be publicly available through the use of data servers after publication, data repositories (i.e., GitHub), and federal data repositories (NCBI and SRA). Funds from this project will also be used to provide open access to all future publications to make them freely available to the public. The research conducted under this project (completed by the PI, undergraduates, and postdoctoral researcher) was presented at two meetings during the first year of the grant. Leisner CP (January 2023) Exploring iridoid compounds in blueberry for human health benefits. Southeastern Fruit and Vegetable Conference, Savannah, Georgia. Leisner CP (June 2022) Genomics-enabled plant physiology to understand climate change impacts on plant nutrition. CROPS 2022, HudsonAlpha Institute for Biotechnology, Huntsville, AL. What do you plan to do during the next reporting period to accomplish the goals?We have several plans for the next reporting period. First, we will continue to screen cultivars of blueberry for the presence of monotropein (Objective 1). Additionally, we will analyze the short-read RNA-Sequencing data we have just received for gene discovery (Objective 1). Next, we will submit samples for Iso-Seq to improve our ability to generate full-length transcripts for potential biosynthetic genes (Objective 1). Finally, we will continue work to clone and functionally characterize key genes in the monotropein biosynthetic pathway in blueberry (Objective 2).Additionally, the data from this work will be presented at the American Society of Plant Biology meeting in summer 2023.

Impacts
What was accomplished under these goals? Several experiments have been completed in Year 1. First, we have completed cloning and functional characterization of the ISY gene in blueberry. Subsequently, we performed mutual rank and other co-expression analyses to determine the role of transcript expression on the production of monotropein blueberry. This work is submitted as a peer-reviewed manuscript and is currently in revision. We are now working on cloning another gene in the pathway, GES, which is a key step differentiation the production of monoterpenes and iridoid compounds (Objective 2). This work was initiated by an undergraduate student in the laboratory under the supervision of the postdoctoral researcher and is now being continued by the postdoctoral researcher in the laboratory. Additionally, the postdoctoral has screened over 20 additional blueberry fruit and leaf samples for the presence/absence of monotropein (Objective 1). These include the new cultivars we proposed to screen in the grant proposal. Interestingly, the rabbiteye ecotype of blueberry seems to produce monotropein in most of the cultivars we survey, which may be a potential new strategy for breeding. Additionally, a wildVaccinium accession(V. darrowii) has been shown to produce iridoids. This is of note since there is now a publicly available genome forV. darrowii. This will help immensely in the gene discovery process. The postdoctoral researcher has also submitted samples for RNA-Sequencing (short-read) to aid in gene discovery and is currently working on preparing samples for long-read RNA-Sequencing (Iso-Seq) that will not require transcriptome assembly and hopefully derive full-length transcript information for cultivars we do not have a genome available for (Objective 1). Additionally, total phenolic content, total anthocyanin, and total flavonoid content were also screened in the same diversity panel used for the iridoid analysis. Quantification of these metabolites was done for 84 members of a blueberry diversity panel and published in 2022 (Rossi et al., 2022). This work was tangential to the grant objectives but served to increase our understanding of the biosynthesis of metabolitesin blueberry with human health benefits.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Edger PP, Iorizzo M, Bassil NV, Benevenuto J, Ferrao F, Giongo L, Hummer K, Leisner CP, Lawas LMF, Li C, Munoz P, et al., (2022) There and back again; historical perspective and future directions for Vaccinium breeding and research studies. Horticulture Research 9: uhac083.
• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Lawas LMF, Kamileen MO, Buell CR, OConnor SE, Leisner CP (In Revision) Transcriptome-based identification of genes involved in monotropein biosynthesis and functional characterization of iridoid synthase in blueberry. Plant Direct.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Rossi G, Woods FM, Leisner CP (2022) Determination of total phenolic, flavonoid and anthocyanin content in a diversity panel of blueberry. HortScience 57(8): 901-909.