Source: CORNELL UNIVERSITY submitted to
INVESTIGATING THE BIOSYNTHESIS OF RESIN GLYCOSIDES IN SWEET POTATO USING NATURAL VARIATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
Reporting Frequency
Annual
Accession No.
1030288
Grant No.
2023-67013-39630
Cumulative Award Amt.
$647,000.00
Proposal No.
2022-08451
Multistate No.
(N/A)
Project Start Date
Jun 1, 2023
Project End Date
May 31, 2026
Grant Year
2023
Program Code
[A1103]- Foundational Knowledge of Plant Products
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
(N/A)
Non Technical Summary
Sweet potato is a highly nutritious crop with an annual US cultivation of over 3 billionpounds. Its different cultivars are rich in starch as well as micronutrients such as vitamins,minerals, carotenoids, flavonoids and anthocyanins, which make it very appealing as a staple crop,in various processed foods such as purees, juices, pies, cakes, noodles and chips, in the foodcolorant industry, as well as in health foods owing to its strong antioxidant potential. Its globalmarket thus is huge (est. >$40 billion), with the crop contributing ~$700 million to the US economy alone. Sweet potato is highly drought resistant and is known as a "crop that surviveswhen other crops fail", even surviving bouts of flooding that damage other crops. This climate resiliency makes sweet potato a centerpiece of hunger elimination and nutritional equality effortsglobally, adding to its value to humanity. However, this crop is threatened by various pests anddiseases such as weevils, wireworm, flea beetles, root rot nematodes as well as various fungi,bacteria and viruses, requiring heavy use of pesticides in major production areas. Due tochanging climate and species ranges of pests and pathogens, identifying how sweet potato can besustainably safeguarded against these biotic challenges is a critical challenge.In this research, we focus on a "hyper diverse" class of defense metabolites called resinglycosides (RGs) that are unique to sweet potato and its parent family Convolvulaceae (morningglories). This family contains many popular horticultural species as well as devastating weeds suchas bindweeds, all of which produce RGs. Substantial prior work, indigenous knowledge and ourpreliminary results suggest that RGs can be effective general deterrence against damage by insects,nematodes as well as fungi and viruses. Our recent workdefined substantial RG diversitywithin and between species, however, the biosynthetic pathway of RGs is completely unknown.Knowing the biosynthetic enzymes will not only open up new genetic targets for breeding of pest-resistant sweet potato varieties but also enable engineering of this trait in other crops such astomato. Our Preliminary Results have highlighted multiple enzyme candidates. In this research, we will (Obj. 1) assess the natural variation of resin glycosides in the USDA sweet potato germplasmcollection (Obj. 2) Identify additional RG biosynthetic candidate genes using RNA-seq and proteomics and (Obj. 3)Validate candidate genes in resin glycoside biosynthesis using functional genomics.Obtaining this foundational knowledgewill pave the way for molecular breeding and crop engineering. Wewill also share results of this research with major breeding centers in North Carolina, Louisianaand other states for translating the findings to the field.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2061450100090%
2062300100010%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1450 - Sweet potato; 2300 - Weeds;

Field Of Science
1000 - Biochemistry and biophysics;
Goals / Objectives
Sweet potato is among the top 10 most cultivated crops globally and is valuable for not just avariety of food products but also for hunger elimination efforts worldwide. Despite its yieldbeing affected by numerous pests and pathogens, its defense metabolites are poorly understood.Here we focus on a hyperdiverse class of its defense compounds called resin glycosides (RGs).Despite its vast structural diversity and known pesticidal/herbicidal activities, the core RGbiosynthetic enzymes are completely unknown. We recently developed metabolomic,computational and functional genomic tools to probe RG diversity and characterize itsbiosynthesis, which identified multiple candidate genes. In proposed research, we will (Objective1)apply these tools to survey 55 diverse accessions from the USDA sweet potato germplasmcollection and associate RG levels with their previously scored insect/nematode susceptibilities.(Objective2) To prioritize genes for functional validation, we will perform RNA-seq from high- andlow-RG cultivars as well as proteomics from sweet potato roots. (Objective3) Role of predictedcandidates in RG biosynthesis will be validated through in vitro and in vivo assays, including viapathway reconstruction in Nicotiana benthamiana. These experiments will identify high-RGlines for sweet potato breeding, generate hypotheses about RG relationship with pestsusceptibility, and enable reconstitution of this pathwayin other crops. Foundational knowledgeof this metabolite class with herbicidal/pesticidal activities will contribute to discoveries tosafeguard sweet potato and the long-term demand for sweet potato-based products.
Project Methods
Obj. 1: Assess the natural variation of resin glycosides in the USDA sweet potato germplasmcollectionUsing semi-targeted liquid chromatography mass spectrometry (LC-MS),we will assess the types and constitutive levels of RGs as well as other defense metabolites among55 accessions available in the USDA germplasm repository that have been previously scored fortheir insect/nematode sensitivity and population structure. This sampling will identify high- andlow- RG accessions, and associate RG levels to their previously known pest susceptibilities.Obj. 2: Identify additional RG biosynthetic candidate genes using RNA-seq and proteomicsUsing three high- and three low-RG accessions, we will identify candidate genes involved in RGbiosynthesis and transcriptional regulation using RNA-seq. RNA-seq in the RG-producing,horticulturally popular species Dichondra argentea Silver Falls and proteomics in sweet potatowill also assist identification of additional candidates.Obj. 3: Validate candidate genes in resin glycoside biosynthesis using functional genomicsOur Preliminary Results highlight multiple candidate RG enzymes, which - along with any otherenzymes identified from Obj. 2 - will be validated using enzyme assays and transientoverexpression/silencing in Ipomoeas, for which we have developed the technical foundation. These enzymes will further be over-expressed in low RG sweet potato accessions and throughpathway reconstruction in Nicotiana benthamiana to determine feasibility of expressing thischemical trait in other crop species.

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