Source: UNIVERSITY OF FLORIDA submitted to
STRUCTURAL ANALYSIS OF THE FLAVONOID PATHWAY TO ENABLE TAILORED PRODUCTION OF BIO-ACTIVE COMPOUNDS IN GRASSES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030349
Grant No.
2023-67013-39629
Cumulative Award Amt.
$650,000.00
Proposal No.
2022-08450
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
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
Flavonoids are compounds plants produce to protect themselves against various stresses that are expected to become more prominent as the result of climate change. This includes attack by pathogenic microbes, temperature extremes, and UV radiation. Several flavonoids are also of interest as anti-cancer drugs or as nutraceuticals - compounds in our food that have positive effects on health, for example by slowing down the effects of aging or reducing the risk of obesity. Of special interest with respect to the latter two applications are 3-deoxyanthocyanidins (3-DOA), which the ceral crop sorghum, but not the other cereals (e.g. maize, wheat), can synthesize, but whose biosynthesis is not yet fully understood. We propose a detailed analysis of how sorghum plants produce flavonoids, as the basis for the tailored production of bio-active compounds, i.e. plants that produce specific flavonoids, and in greater quantity. This approach relies on a detailed analysis of the enzymes involved in flavonoid biosynthesis to figure out their exact mode of action. As a next step, their properties can be modified.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2061520104030%
2061520100050%
2061520108010%
2061520102010%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1520 - Grain sorghum;

Field Of Science
1040 - Molecular biology; 1000 - Biochemistry and biophysics; 1080 - Genetics; 1020 - Physiology;
Goals / Objectives
To obtain recombinant sorghum flavonoid biosynthetic enzymes and determine their activity and kinetic parametersTo determine the 3D-structure of sorghum flavonoid biosynthetic enzymes based on X-ray crystallographyTo determine the expression of flavonoid biosynthetic genes, with a focus on 3-deoxyanthocyanidins, using sorghum and maize in tissue culture.
Project Methods
1. Generation of recombinant flavonoid biosyntehtic enzymes in E. coli with the use of expression vectors. Purification of the recombinant enzymes using chromatography2. Development of structural models of flavonoid biosynthetic enzymes based on X-ray crystallography of purified recombinant enzymes in crystalline form.3. Kinetic analyses to determine substrate specificity, affinity, effect of inhibitors4. Development of catalytic models based on 2 and 3 above and validation based on site-directed mutagenesis of amino acid residues predicted to be involved in catalysis.5. Determine the expression of flavonoid biosynthetic genes in regenerating sorghum leaf whorl explants using transcriptome profiling (RNA-seq), with a focus on the genes involved in the biosynthesis of 3-deoxyanthocyanidins6. Transgenic validation of the candidate genes identified in 5 above with the use of RNA interference to reduce the expression level of candidate genes. This is expected to result in a reduction in the amount of 3-deoxyanthocyanidin that are being produced in tissue culture.7. Generation of transgenic Black Mexican Sweet cell lines that have the ability to expression 3-deoxyanthocyanidins.

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

Outputs
Target Audience:1. Biochemists, molecular biologists and geneticists focused on flavonoid biosynthesis in sorghum and related grasses 2. Food scientists and biomedical scientists interested in flavonoids as health-promoting compounds Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training and educational opportunities for several students and professionals: Kang: Training of three graduate students (Jacob Lewis, Punia Neetika, Min Wang) and four undergraduate students (Ashley Weller, Molly Morris, Madali Madilyn, Alexandria Loeb) Sattler: training of one graduate student (Yuvraj Chopra) Vermerris: training of one graduate student (Saddie Vela) and two research assistants (Alyssa Davis, Dominic Maglione). How have the results been disseminated to communities of interest?We have published two peer-reviewed journal publications What do you plan to do during the next reporting period to accomplish the goals?Objectives 1, 2: Activity assays and determination of the 3D-structure for the following enzymes F3'H, F3',5'H, FNSII, ANS, FLS, FNR, UFGT Objective 3: Complete the data analysis to identify differentially expressed genes in brown vs. beige callus to identify genes involved in the production of 3-DOA and their precursors. Clone the corresponding cDNA's for expression of sorghum enzymes in E. coli and Black mexican Sweet maize callus.

Impacts
What was accomplished under these goals? 1. Sorghum cDNA's encoding the enzymes listed below were cloned in expression vectors that were subsequently introduced in E. coli to obtain purified recombinant protein: Chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavonoid 3' hydroxylase (F3'H), flavonoid 3', 5' hydroxylase (F3'5'H), flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), anthocyanin synthase (ANS), anthocyanidin reductase (ANR), flavonol synthase (FLS), flavanone 4-reductase (FNR), flavone synthase (FNSII), UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT) The activity and kinetic parameters of the following enzymes was determined: CHS, CHI/CHIL, DFR, FNR, FNR 2. The 3D-structure of the following enzymes was completed and published: CHS, CHI, CHIL, DFR, FLS, ANR In preparation for the determination of the 3D structure, the crystallization of the following enzymes has been initiated: F3'H, F3',5'H, FNSII, F3H, ANS, FLS, ANR, UFGT 3. Leaf whorl explants of sorghum inbred lines RTx430 and P898012 were placed on callus induction medium (CIM) with and without polyvinylpyrrolidone (PVP). The explants were propagated until callus started to form, and the clumps of callus were transferred to fresh CIM (with or without PVP) every two weeks. After 6-12 weeks brown and beige callus started to form. The brown callus produces 3-deoxyanthocyanidins (3-DOA). Brown and beige callus was collected from individual culture plates, frozen in liquid nitrogen and used for RNA extraction and subsequent RNA-sequencing (at Novogene). A total of 24 libraries (twelve RTx430 (6 beige, 6 brown), twelve P898012 (6 beige, 6 brown)) were sequenced and the data have been released. The data will be used to identify differentially expressed genes in brown vs. beige callus, which is expected to result in the identification of genes involved in the production of 3-DOA and their precursors.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Lewis JA, Zhang B, Harza R, Palmer N, Sarath G, Sattler SE, Twigg P, Vermerris W, Kang C. (2023) Structural Similarities and overlapping activities among dihydroflavonol 4-reductase, flavanone 4-reductase, and anthocyanidin reductase offer metabolic flexibility in the flavonoid pathway. International Journal of Molecular Sciences 24(18):13901. https://doi.org/10.3390/ijms241813901
  • Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Lewis JA, Jacobo EP, Palmer N, Vermerris W, Sattler SE, Brozik JA, Sarath G, Kang CH (2024) Structural and interactional analysis of the flavonoid pathway proteins: Chalcone synthase, chalcone isomerase and chalcone isomerase-like protein. International Journal of Molecular Sciences 10.3390/ijms25115651