Progress 06/01/24 to 05/31/25
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?Vermerris: Training of one graduate student (Saddie Vela), one post-doc (Timothy Changa), and three undergraduate research assistants (Dominic Maglione, Hayden Seymour, Genevieve Bell). Kang: Training of three graduate students (Jacob Lewis, Punia Neetika, Min Wang) and seven undergraduate students (Ashley Weller, Molly Morris, Madali Madilyn, Alexis Maguffee, Gray Galio, Timothy Chapman, Wyatt Mintken) Sattler: Training of one graduate student (FTE 1.0 technical; Yuvraj Chopra) and undergrad Fin Rail. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Objectives 1, 2: Complete purification, perform activity assays and determine the 3D-structure for the following enzymes F3'H, F3',5'H, FNSII, ANS, FLS, FNR, UFGT, CPR. Determine binding affinity for pairs of enzymes to investigate their role in the formation of metabolons. Objective 3: Validate the candidate genes involved in the production of 3-DOA by determining if maize BMS callus produces 3-DOA following the expression of individual candidate genes in combination with a Myb transcription factor that induces the production of the substrate molecules. Determine enzymatic activity of purified recombinant enzymes encoded by the three candidate genes. Structural analysis of purified recombinant protein.
Impacts
What was accomplished under these goals?
Objective 1: During Year 2 of the project, 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: flavanone 3-dioxygenase (F3H), three isozymes of cytochrome P450 reductases (CPR), 4-coumarate CoA ligase (4CL; Brown midrib2). In addition, as a continuation of experiments initiated in Year 1 of the project, expression conditions for the following recombinant sorghum enzymes were optimized and the enzymes were purified: Chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL) , dihydroflavonol reductase (DFR), anthocyanin synthase (ANS), anthocyanidin reductase (ANR), flavonol synthase (FLS), flavanone 4-reductase (FNR), UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT). We have been modifying the sequence for three cytochrome P450s (flavone synthase (FNSII), flavonoid 3', 5' hydroxylase (F3'5'H) and flavonoid 3' hydroxylase (F3'H) to increase the expression level. We have been modifying the sequences of (4CL, ANR, F3H) to improve crystal formation. Objective 2: The 3D-structure of the following enzymes was completed: 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, FNR, UFGT. We have begun to investigate the potential interaction among the above-listed enzymes in metabolons. Current evidence supports the hypothesis that an organized nanoparticulate assembly of enzymes enables an efficient biosynthetic pathway for monolignols and flavonoids. These supramolecular complexes of different enzymes organized through non-covalent interactions, create stable complexes or nanostructures that can change shape or size upon a chemical cue. Moreover, the chemical machinery of these nanostructures tightly links the flavonoid biosynthetic pathway with the monolignol biosynthetic pathway through the common intermediate p-coumaroyl CoA. Thus, the enzymes-complexes in those two biosynthetic pathways hold promise as targets for the improvement of crops, by modulating the flux between these two agronomically important pathways. Objective 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. Based on our prior published research, 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. A total of 24 libraries (twelve RTx430 (6 beige, 6 brown), twelve P898012 (6 beige, 6 brown)) were sequenced and the data have been analyzed. The data were used to catalog differentially expressed genes in brown vs. beige callus. Three candidate genes involved in the production of 3-DOA were identified among the differentially expressed genes based on sequence similarity with sorghum genes encoding anthocyanidin synthase, and absence of orthologous sequences in maize (which cannot produce 3-DOA). The cDNA's for the three candidate genes have been cloned in bacterial expression vectors for structural characterization and enzyme activity assays. Two of the three cDNA's have also been cloned in plant expression vectors to be used for the transformation of Black Mexican Sweet (BMS) maize callus. This will enable us to validate candidate gene function.
Publications
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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
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