Progress 06/01/24 to 05/31/25

Outputs
Target Audience:The target audience for this project in the reporting period was the phytochemistry research community. PI Moghe presented work from this grant at the Plant and Animal Genome meeting in January, 2025. The talk was titled "Unraveling the Hidden Diversity and Biosynthesis of Resin Glycoside Acylsugars in the Morning Glory Family" and was presented in the session "Genomics of Medicinal and Aromatic Plants". The grad student and postdoc also presented this research in two forums. We also discussed the methods involved in this research (LC-MS) in two publications: 1)Analysis of plant metabolomics data using identification-free approaches (published in Applications in Plant Sciences) and 2)Extraction, Annotation, and Purification of Resin Glycosides from the Morning Glory Family (Convolvulaceae) (published in Methods in Molecular Biology). Changes/Problems:OBJECTIVE 1 Lack of Clustering by Resistance Trait: It was expected that accessions labeled as pest-resistant would cluster together based on similar resin glycoside profiles, but in our preliminary assessment with 10 varieties, they did not. This suggests that resistance may not be solely driven by RG composition and could involve other biochemical pathways or gene regulatory mechanisms--highlighting the multifactorial nature of defense traits. We are still validating our results with more consistent LC-MS with more varieties to solidify this inference. Problems with plant establishment: The sweet potato accessions received from the USDA GRIN repository arrive either as cuttings or in culture tubes. While the cuttings typically establish well, the accessions provided in culture tubes are much more challenging to maintain. These often suffer from wilting and do not survive the acclimatization process. Additionally, transitioning these plants from sterile in vitro conditions to soil involves a labor-intensive protocol. Establishing them first in sterile substrate and then gradually transferring them to greenhouse conditions is not only delicate but also time-consuming, often requiring over two month before plants are fully stabilized and ready for making replicate cuttings. OBJECTIVE 2 RNA-seq and proteomics:Given lack of good quality genomes in this family, limited inferences can be derived from RNA-seq and proteomics experiments. Nonetheless, we were able to obtaingood candidates that we are currently pursuing. OBJECTIVE 3 Novel enzyme candidates: We also obtained novel candidates for fucose synthase in our system. VIGS was performed and 4/5 lines for each of the two showed reduction in RGs. These results will be further verified in the coming months with additional replicates and in vivo assays.Even though we can see P450 activity, identifying the position of hydroxylation is challenging because the 11th position hydroxylation is a critical signature for RGs. We are exploring alternative ways of validating this observation. In vivo validation: We faced problems with Ipomoea nil seed germination rates, bottlenecking our VIGS validation assays. We therefore developed a hairy root system that may potentially address this validation challenge. We are also developing the Nicotiana benthamiana system for faster validation of initial pathway activities. What opportunities for training and professional development has the project provided? One graduate student, one postdoc and two undergraduates worked on this project during the reporting period. The trainees played a role in literature review, experimentation and experimental analyses. The graduate student wrote an internal Cornell grant proposal for an allied RG project - to determine where RG are produced in sweet potato roots. This project - an extension of the NIFA grant - was successfully funded. The activity gave the student an opportunity to develop their research idea, build collaborations and write a proposal. The postdoctoral associate Dr. Irfan attended the6th Annual North American Mass Spectrometry Summer School, Madison, Wisconsin, USA (July 8-11, 2024) to improve his understanding of mass spectrometry techniques required for this research. How have the results been disseminated to communities of interest?The target audience for this project in the reporting period was the phytochemistry research community. PI Moghe presented work from this grant at the Plant and Animal Genome meeting in January, 2025. The talk was titled "Unraveling the Hidden Diversity and Biosynthesis of Resin Glycoside Acylsugars in the Morning Glory Family" and was presented in the session "Genomics of Medicinal and Aromatic Plants". Bhaswati Sarmah gave a lightning talk on this project in the Plant Breeding and Genetics student seminar series, titled "Exploring Resin Glycoside Diversity in Sweet Potatoes for Protection Against Biotic Stress" Dr. Mohammad Irfan presented a talk titled "Elucidating the biosynthesis and structural diversity of resin glycosides of morning glory family" at the6th Annual North American Mass Spectrometry Summer School, Madison, Wisconsin, USA (July 8-11, 2024) We also discussed the methods involved in this research (LC-MS) in two publications: 1)Analysis of plant metabolomics data using identification-free approaches (published in Applications in Plant Sciences) and 2)Extraction, Annotation, and Purification of Resin Glycosides from the Morning Glory Family (Convolvulaceae) (published in Methods in Molecular Biology). What do you plan to do during the next reporting period to accomplish the goals?OBJECTIVE 1 We will not be able to complete assaying all 55 accessions as proposed because many accessions are not available in the USDA sweet potato germplasm, and because of lack of enough personnel to subculture and send us slips/explants. We will, however, have finished 40-45 accessions by next reporting period. We will have completed 25 by end of coming June. We will have more concrete idea of the cell types producing resin glycosides through a planned microdissectione experiment in collaboration with Mike Scanlon lab at Cornell. Results from most of the samplings will have been analyzed and readied for publication. OBJECTIVE 2 Objective 2 is completed and no specific experiments are planned for the next reporting period OBJECTIVE 3 We will have identified UGTs and BAHDs in RG biosynthesis by next reporting period It is possible that most of the pathway will have been characterized by then, with some additional experiments remaining We plan to deploy the hairy root system for faster validation of gene candidates in the near future. We expect to have first draft of the publication by the next reporting period - separate from the diversity analysis paper.

Impacts
What was accomplished under these goals? OBJECTIVE 1 Optimizing Sampling Time for RG Extraction A time-course study was conducted using four genetically diverse sweet potato accessions (308200, 634375, 634512, and 564128), with sampling at 2, 3, 4, and 5 months after planting.Resin glycoside (RG) levels were lowest at 2 months, indicating limited metabolite accumulation at early developmental stages.Month 5 showed variable trends depending on the genotype, with some accessions retaining high RG levels and others exhibiting a decline.Month 3 consistently showed the highest RG abundance across most accessions, establishing it as the ideal time point for metabolomic and transcriptomic analysis.Hierarchical clustering of metabolomic data confirmed both temporal and accession-specific patterns, highlighting the interplay between development and genetic background in RG biosynthesis.Based on these, Month 3 was chosen as the optimal sampling point because it consistently showed the highest resin glycoside accumulation across all accessions, indicating peak biosynthetic activity. This timing maximizes the likelihood of capturing meaningful metabolomic signals for downstream analysis. Characterization of RG Structural Components Detailed LC-MS-based profiling was performed to analyze the chemical structures of RGs, focusing on their acyl, hydroxyacyl, and sugar components.Major acyl chains dominant in sweet potato are: decanoic, cinnamic, crotonic, and dodecanoic acids, contributing to structural variation.Hydroxyacyl units such as C16OH and C16OHOH were commonly observed, indicating conserved lipid modifications across accessions.The glycosylation profile frequently featured hexose and deoxyhexose sugars, suggesting shared biosynthetic routes despite varietal differences.These findings suggest that resin glycosides in sweet potato share conserved core structural features--particularly in their hydroxyacyl and sugar components--across diverse accessions, indicating a common biosynthetic origin. However, variation in acyl chain composition, including the presence of decanoic, cinnamic, and crotonic acids, points to biochemical diversification that may influence the biological activity or ecological function of RGs in different genotypes. Spatial Localization of RGs in Sweet Potato Tissues To investigate the specific regions of the storage root where RGs are produced or accumulate, sequential peel layers were extracted and analyzed individually via LC-MS.High RG abundance was consistently detected in the outermost layers (Peels 1-3), which correspond to the periderm and suberized protective tissues like phellem and phellogen.Inner tissue layers showed significantly lower RG content, reinforcing the hypothesis that RG biosynthesis is primarily localized to the epidermal and subepidermal regions involved in defense.These results indicate that resin glycoside biosynthesis or accumulation is spatially confined to the outer protective tissues of the sweet potato root, particularly the periderm. This localization supports their role as frontline defense metabolites, concentrated where exposure to pests and environmental stress is greatest. RG Diversity Across Accessions We extracted the resin glycosides from 19 diverse accessions till date.Accessions generally cluster according to their geographic origin or population group. For instance, accessions from the Far East (e.g., PI 666137 and PI 585077) are closely grouped, indicating a possible shared ancestry or similar metabolite profiles.Accessions from North American-1 (PI 634411 and PI 566612) and North American-2-1 accessions (PI 566628 and PI 666136), form a separate cluster, highlighting sub-population divergence.Accessions with reported pest resistance (PR) such as PI 666137 and PI 666136 do not cluster together, suggesting that resistance to pests may arise from different underlying biochemical or genetic mechanisms, not necessarily reflected in the clustering pattern.PI 308200, from the Pacific Islands, appears relatively isolated in the cluster. This could indicate a unique RG biosynthetic profile, consistent with its notably lower RG production across developmental stages as seen in earlier analyses.The observed clustering pattern seems to reflect sample-level biochemical or gene expression differences more than shared ancestry or resistance traits, emphasizing the complexity of metabolite regulation in sweet potato.The clustering of resin glycoside profiles among diverse sweet potato accessions reveals that metabolite composition is influenced more by biochemical or gene expression variation than by geographic origin or resistance traits alone. This suggests that pest resistance may evolve through distinct metabolic pathways across genotypes, and highlights the complexity of specialized metabolite regulation in sweet potato populations. OBJECTIVE 2 RNA-seq was not done as planned because in Year 1 we had already done time-series and TFA-noTFA RNA-seq We performed proteomics from extracted Golgi compartments from Ipomoea nil leaves Proteomic results pointed to additional candidate genes for RG biosynthesis OBJECTIVE 3 Size exclusion chromatography was performed from roots of Ipomoea nil, Ipomoea tricolor and Dichondra argentea. An assay system was developed for purification and analysis of UGTs. From the exclusion fraction, we were able to detect definite UGT activity that tracked with presence/absence of UGTs across the different tissues. With the successful development of the UGT assay, we cloned 21 UGT candidates in expression vectors and are currently expressing them in Nicotiana benthamiana leaves to detect products. Cytochrome P450 activity was detected from Ipomoea nil and Dichondra argentea, but we could not validate the position of hydroxylation using LC-MS. We performed these assays using C13-labeled substrates, and we were able to detect activities for both C16 and C14 fatty acids. Hairy root transformation was tested in several members of morning glory family such as Ipomea nil, Ipomoea tricolor, Ipomoea batatas (sweet potato). For that, 35S:GUS vector was transformed using Agrobacterium rhizogenes mediated transformation. We were successful in obtaining hairy roots from Ipomoea nil leaves.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Yuan X, Smith SS, Moghe GD (2025) Analysis of plant metabolomics data using identification-free approaches. Applications in Plant Sciences, e70001

Progress 06/01/23 to 05/31/24

Outputs
Target Audience:The target audience for this project in the reporting period is the phytochemistry research community. Preliminary work from this grant was presented briefly at one campus invited talk by the PI. The postdoctoral associate (Dr. Mohammad Irfan) presented his results in detail as a presentation in the Plant BiologySection retreat at Cornell University, and as a poster at the ASPB Northeast Section Meeting in April 2024, where he was awarded prize for the Second Best Poster. Changes/Problems:It took us a little more time than expected to standardize the growth protocol of sweet potato transplants. We expected Aim 1 to be done by mid-Year 2, but it may get delayed due to limited greenhouse space. Nonetheless, a vast majority of the accessions will be screened by end of Year 2. What opportunities for training and professional development has the project provided?One postdoc, one graduate student, one undergraduate student worked on this project. An additional MS student worked part-time on this project during summer 2023. The postdoc is a first-generation PhD degree holder. The graduate student attended an LC-MS training workshop at UC-Davis West Coast Metabolomics Center to learn how to analyze LC-MS data. The undergraduate is a freshman and working in our lab was his first research experience. How have the results been disseminated to communities of interest?Through seminars, website and publications (accepted) as highlighted in above sections. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Complete RG analysis for ~40 accessions. Make associations with insect susceptibility. Start drafting manuscript. Objective 2: I anticipate this goal will be completed by end of next reporting period. We will have candidates from RNA-seq and proteomics experiments. Objective 3: We will have confirmed and published BAHD activity. We hope to be in drafting stages of another enzyme activity for another manuscript.

Impacts
What was accomplished under these goals? Objective 1: We established a protocol for growing sweet potatoes from transplants and initiated screening of the accessions. We identified 55 accessions as proposed and so far 21 accessions have been obtained from the Sweet Potato Germplasm Center in a stepwise fashion. All plants are in different stages of growth. Preliminary time-series LC-MS analyses were performed at 2,3,4,5 months after transplanting to determine when RGs are produced, revealing RG presence at all stages but increased diversity at 3 and 4 months. Sampling and data analyses for all 23 accessions will be completed by end of summer. Objective 2: We performed RNA-seq from Dichondra time-series samples as well as Ipomoea nil, with and without trifluoroacetic acid (TFA) spray. We found TFA spray increased RG levels, and therefore we expect RG biosynthetic genes to be upregulated upon TFA spray. Multiple candidate genes from different enzyme families were obtained from time-series and TFA datasets. Candidate gene selection will be completed within the next 2 months. Proteomics experiments are being planned. Objective 3: We confirmed in vitro activity of our BAHD candidate (BAHD5) using further assays. Six additional BAHDs were cloned and purified. VIGS of the BAHD5 candidate in Ipomoea nil is underway.

Publications

• Type: Book Chapters Status: Accepted Year Published: 2024 Citation: Lars H. Kruse*, Alexandra A. Bennett, Vishwa J. Baruah, Mohammad Irfan, and Gaurav D. Moghe (accepted) Extraction, annotation, and purification of resin glycosides from the morning glory family (Convolvulaceae), in Chapter Natural Product Isolation and Identification by chromatography and spectroscopy, Methods in Molecular Biology