Progress 12/01/19 to 11/30/20
Outputs
Target Audience:The target audience reached by the efforts performed as part of this project included undergraduate and graduate students, postdocs, faculty and also industry representatives. The various target audiences were reached through lectures and posters given by PI Grotewold, the postdocandthe graduate student participating in the project. Visiting scholars from various countries hosted by the PI's laboratory have been exposed to and participated in different aspects of the project. Changes/Problems:We did encounter a few challenges that delayed some of the experiments. COVID caused certainly a major disruption, but we are now starting to catch-up from those delays. Another unexpected delay resulted for how long the generation of the transgenic lines took at theWisconsin Crop Innovation Center - it took almost a year longer than planned. But we have all the plants now and we will continue with the analysis. What opportunities for training and professional development has the project provided?The project has continued to enhance the expertise ofvisiting scholar Oscar Castaneda Mendez in tomato research, an are of investigation that only became possible in my lab thanks to this grant. He tried to get into graduate school last year, but did not succeed and will apply this year again. Lina Gomez Cano, who also started as a visiting scholar in the lab, was accepted in 2019 as a graduate student in thedual graduate program between Molecular Plant Sciences (MPS) and Biochemistry & Molecular Biology at MSU, and completed her first year in the program, publishing a first author paper (see publications resulting from funding). In addition, Dr. Nan Jiang, who also participated in the project and was responsible for all the field work, is starting to apply to faculty positions. How have the results been disseminated to communities of interest?The PI has given presentations at meeting and seminars at other universities. In addition, Dr. Nan Jiang was invited to give an oral presentation at ASPB 2020, and while his presentation focused on a project only partially related to this, the findings have significant implications for understanding how flavonoids are regulated. What do you plan to do during the next reporting period to accomplish the goals?This completes the third year of funding, hence experiments will continue but not supported by this project.
Impacts
What was accomplished under these goals?
Objective 1: We have continued to investigate the finding that maysin accumulates in leaves of young plants. These high levels of maysin (which are highly influenced by genotype) decrease significantly after three weeks of growth of the plants. Different from what happens in silks, where maysin accumulation is controlled by the R2R3-MYB transcription factor P1, P1 appears to have a minor contribution in leaves and we have identified another candidate transcription factor likely to be responsible for most of the function. It is puzzling why P1 has even a minor contribution, as its expression in leaves in undetectable. We are also exploring the possibility that a genetic factor linked to P1, but not P1 itself, could be responsible. As part of a cooperation with the Wisconsin Crop Innovation Center, CRISPR-Cas9 was used to knock-out three candidate enzymes (annotated as UDP-glycosyl transferases, or UGTs) that are regulated by P1 in pericarps and silks. The T1 seeds (generated in the Hi-II background) were received in September 2020, and are currently being planted in the greenhouse for genotyping and crossing. Unexpectedly, by characterizing a transposon Mu insertion in UGT (GRMZM2G383404), we determined that the mutant also has a weak salmon silk phenotype, but continues to accumulate maysin. This would be the first mutant in which we found the co-existance in the silks of 3-deoxyanthocyanidins (responsible for thesalmon silkphenotype) and maysin. Objective 2: While some of the mutants described above are possibly involved in the formation of maysin, it is very possible that the others participate in the formation ofthe other flavones present in maize pericarps and silks. These candidate genes are being expressed in yeast to determine which substrates they prefer - we anticipate results from such experiments later in the year. Objective 3: We have introduced maize FNS1 and a Gentiana C-glycoysl transferase into two genotypes of tomato (MP1 and LH82) under the control of the constitutive CH1 promoter, or the glandular trichome-specific pMKS1 promoter. Plants expressing each of the transgenes have been validated and the F1 cross is now being assayed for the accumulation of various flavones, both in the fruits as well as in the leaves.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Gomez-Cano, L., Gomez-Cano, F., Dillon, F.M., Alers-Velazquez, R., Doseff, A.I., Grotewold, E., and Gray, J. (2020) Discovery of modules involved in the biosynthesis and regulation of maize phenolic compounds. Plant Sci, 291: 110364.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Jiang, N., Dillon, F.M., Silva, A., Gomez-Cano, L., and Grotewold, E. (2020) Rhamnose in plants From biosynthesis to diverse functions. Plant Sci, In Press
|
Progress 12/01/17 to 11/30/20
Outputs
Target Audience:The target audience reached by the efforts performed as part of this project included undergraduate and graduate students, postdocs, faculty and also industry representatives. The various target audiences were reached through lectures and posters given by PI Grotewold, the postdoc and the graduate student participating in the project. Visiting scholars from various countries hosted by the PI's laboratory have been exposed to, and participated, in different aspects of the project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has continued to enhance the expertise of visiting scholar Oscar Castaneda Mendez in tomato research, an area of investigation that only became possible in my lab thanks to this grant. He tried to get into graduate school last year, but did not succeed and is applying this year again. Lina Gomez Cano, who also started as a visiting scholar in the lab, was accepted in 2019 as a graduate student in the dual graduate program between Molecular Plant Sciences (MPS) and Biochemistry & Molecular Biology at MSU, and completed her first year in the program, publishing a first author paper (see publications resulting from funding). In addition, Dr. Nan Jiang, who also participated in the project and was responsible for all the field work, is starting to apply to faculty positions. How have the results been disseminated to communities of interest?The PI has given presentations at scientific conferences and seminars at other universities. In addition, Dr. Nan Jiang was invited to give an oral presentation at ASPB 2020, and while his presentation focused on a project only partially related to this, the findings have significant implications for understanding how flavonoids are regulated. What do you plan to do during the next reporting period to accomplish the goals?This is the final report.
Impacts
What was accomplished under these goals?
Objective 1: We have been able to combine mutants in a1, sm1 and sm2 in the presence of the dominant P1-rr allele to significantly increase maysin production in kernels. Crosses to the recently knocked-out ZmCGT1 are underway and we will not know the result for some time. Objective 2: We have characterized several genes that appear to participate in different aspects of flavone accumulation, in addition to those already identified as part of the pathway. We accomplished this using combinations of computtional approches, expression in yeast as well as transient expression in Nicotiana benthamiana. The specific chemistries that the respective gene products are involved in are currently being determined, since all the information that we currently have derives from liquid chromatography-mass spectrometry (LC-MS), and we are limited by the availability of standards. We have also identified a maysin biosynthetic process in maize young plants, which appears to be controlled independently of teh accumulation of maysin in silks and pericarps. A candidate R2R3-MYB transcription factor (MYB154) is being currently confirmed as being involved in the regulation of this pathway. Objective 3:We have introduced maize FNS1 and a Gentiana C-glycoysl transferase into two genotypes of tomato (MP1 and LH82) under the control of the constitutive CH1 promoter, or the glandular trichome-specific pMKS1 promoter. Plants expressing each of the transgenes have been validated by real-time reverse transcriptase PCR, and the location of the transgene(s) was determined in a few instances by TAIL-PCR. The F1 crosses are now being assayed for the accumulation of various flavones, both in the fruits as well as in the leaves.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Righini, S., Rodriguez, E.J., Berosich, C., Grotewold, E., Casati, P., and Falcone Ferreyra, M.L. (2019). Apigenin produced by maize flavone synthase I and II protects plants against UV-B-induced damage. Plant, Cell & Environment 42, 495-508.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Gomez-Cano, L., Gomez-Cano, F., Dillon, F.M., Alers-Velazquez, R., Doseff, A.I., Grotewold, E., and Gray, J. (2020) Discovery of modules involved in the biosynthesis and regulation of maize phenolic compounds. Plant Sci, 291: 110364.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
119. Jiang, N., Dillon, F.M., Silva, A., Gomez-Cano, L., and Grotewold, E. (2020) Rhamnose in plants From biosynthesis to diverse functions. Plant Sci, 302: 110687
|
Progress 12/01/18 to 11/30/19
Outputs
Target Audience:The target audience reached by the efforts of this project include undergraduate and graduate students, postdocs and faculty. The target audience was reached through lectures, worhops and classes given by PI Grotewold. The laboratory also hosts visiting scholars from several countries that are exposed (and often participate in some aspect) of the project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project resulted in visiting scholar Oscar Castaneda Mendez becoming an expert on tomato work. In fact, he is currently in Israel presenting results from this project at theXVI Solanaceae Conference in Jerusalem, and also at the Volcani Center. I should mention that Oscar had no experience working with tomato prior to starting with this project. Oscar is planning to apply to graduate school this fall. Another visiting scholar, Lina GomezCano, who was involved with the experiments in yeast took a leadership role in profiling the maize genes involved in flavonoid metabolism, and a paper is currently being reviewed. Lina applied and was admitted to the dual graduate program between Molecular Plant Sciences (MPS) and Biochemsitry & Molecular Biology (BMB) this fall and has just started taking classes. A new visiting scholar,Alexander Silva Cordoba, started in April 2019, and has been instrumental in characterizing maysin levels in leaves, and also in working with entomology colleagues at MSU to develop Helicoverpa zea feeding assays to test the bioactivity of many of the compounds taht we are generating in yeast, tomato and maize plants. How have the results been disseminated to communities of interest?So far, the PI has given presentations at meeting and seminars at other universities, and members of the team have presented results at various events, such as theXVI Solanaceae Conference and the Maize Genetics Conference.? What do you plan to do during the next reporting period to accomplish the goals? Finish analyzing pericarps at different developmental stages of a1 sm2 lines Characterize moleculalry and metabolically the CRISPR lines targeting various UGTs Test the new constructs in yeast to determine whether we can succesfully engineer maysin and other flavones Cross the trangenic tomato lines and analyze metabolically the products formed
Impacts
What was accomplished under these goals?
Objective 1: Redirect maize flavonoid biosynthesis towards the formation of health beneficial flavones(Years 1 - 3; Progress 50%): We completed the analysis of the double mutant (a1 sm1 and a1 sm2) silks. We determined that the a1 sm2 progeny segregates in a perfectly Mendelian fashion with regards to high, mid and low maysin, but the a1 sm1 material shows a gradient of maysin levels that have yet to understand. Promising, the a1 sm2 lines that accumulate low levels of maysin showed high accumulation of the flavone luteolin. We have now collected the material from the pericarps in the field and it is being analyzed for flavone levels. Beyond what was originally proposed, we also found that maysin accumulates at very high levels in leaves of young (< 3 weeks old) maize plants. The level of maysin can reach 0.1% of the leaf dry weight. We are in the process to understand what this means, but much to our surprise, we found that sm1 mutant plants accumulate instead rhamnosylisoorientin (the expected precursor), or unexpectedly, luteolin, and which compound accuulates depends of which sm1 allele is being investigated. This finding opens a number of opportunities regarding how pathway intermediates are modified, and also what is the role of rhamnose in maize (Sm1 corresponds to a rhamnose synthase). Progress with the CRISPR lines has been very slow becuase of a number of technical and personnel bottlenceks at theWisconsin Crop Innovation Center?. However, we have been assured that we should have transgenic lines available to work with by the end of Spring 2020. Objective2: Identify new maize flavone biosynthesis and modification genes(Year 2; Progress 40%): Complementing our experiments in yeast, we have conducted a genome-wide survey in maize of genes that could be involved in the biosynthesis and metabolism of phenlic compounds. The manuscript is currently under revision. We have also generated a translational fusion of maize F2H and UGT5 to express in yeast in the hope to be able to increase the flux towards the maysin pathway. As an alternate engineering approach, we synthesized a clone expressing Gentian open reading frame for a UDP-glucosyl transferase (GtUF6CGT) reported to be able to directly conjugate through a C-C bond glucose to the flavone apigenin or luetolin. The activity of this protein is currently being tested in the yeast cells harboring several of the maize pathway genes with the objective to enhance maysin production. Objective 3: Engineer maysin production in tomato(Year 2 - 3; Progress 50%): We generated transgenic tomato plants (Genotype M82) expressing ZmF2H1, ZmFNS-1,GtUF6CGT,or SM2 under the constitutive pCHI1 or trichome-specific pMSK1 promoters using Agrobacterium-mediated transformation. The expression of the four transgenes in 3-5 lines for each of the constructs was verified by RT-qPCR. Seeds are currently being bulked-up and we anticipate to start making the crosses to generate lines expressing combination of the transgenes capable of producing maysin from the available precuros eriodyctiol (flvanone) this autumn.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Righini, S., Rodriguez, E.J., Berosich, C., Grotewold, E., Casati, P., and Falcone Ferreyra, M.L. (2019). Apigenin produced by maize flavone synthase I and II protects plants against UV-B-induced damage. Plant, Cell & Environment 42, 495-508.
|
Progress 12/01/17 to 11/30/18
Outputs
Target Audience:Efforts as part of this project include lectures, workshops and classes given by PI Grotewold. The Grotewold lab also hosts visiting scholars from several countries which were exposed to various aspect of the project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A rotation student (Paul Fiesel) had the opportunity to analyze flavone content in the segregating maize lines; this was his first experience working with maize. Two visiting scholars aspiring to become graduate students (Lina Gomez Cano and Oscar Fernando Castaneda Mendez) have been working on this project since it started. Both have acquired knowledge that they did not have before. For example, Lina was trained as a chemist and she has become very proficient in analyzing phenolics by HPLC and LC-MS. Oscar was an agronomis without molecular biology experience and he has cloned and transformed the various genes into tomato. Both of them are supervised by a senior postdoc in the lab, Dr. Nan Jiang, who is an expert on flavonoids and who is acquiring in this way some of the essential leadership skills necessary for an independent position. How have the results been disseminated to communities of interest?So far, the PI has given presentations at meetings and other universities, and the stduents and postdocs have presented at local meetings posters and seminars. What do you plan to do during the next reporting period to accomplish the goals?We will hopefully obtain the maize CRISPed plants that will be characterized and analyzed by Dr. Jiang. We will continue the generation of the transgenic tomato plants with the maysin pathway genes. We will analyze other OMTs and P450s that might participate in flavone biosynthesis in our yeast lines.
Impacts
What was accomplished under these goals?
Objective 1: Redirect maize flavonoid biosynthesis towards the formation of health beneficial flavones(Years 1 - 3; Progress 25%): Progress has been made toward this aim at two levels: We generated double mutants between known and newly-characterized flavone biosynthesis genes, and characterized the segregating F2 populations by LC-MS of silk tissues for the accumulation of glycosylated flavones (e.g., maysin) and aglycones (e.g., luteolin). We found a very clear segregation of low maysin and high luteolin (>1,000 increase) levels some of the double mutants, but the effect was less clear in others. We have bulked-up the material this summer to analyze flavone accumulation in the seed at different developmental stages. We have submitted guide RNA constructs to perform knock-outs of four maize C-glycosyltransferases, which are good candidates for catalyzing flavone C-glycosylation in the silk maysin biosynthesis pathway. These constructs have already been transformed into Agrobacterium by the Wisconsin Crop Innovation Center Plant Transformation Facility, and are expected to go into HiII maize plants in the next month (Nov-Dec., 2018). Objective2: Identify new maize flavone biosynthesis and modification genes(Year 2, Progress 15%):We have continued to reconstitute the flavone pathway in yeast cells harboring the Arabidopsis cytochrome P450 reductase gene (WAT11). WAT11 cells harboring maize pathway genes and fed with naringenin accumulate significant levels of both luteolin and apigenin (both desirable because of their demonstrated nutritional properties). We tested whether BM3 (encoding a O-methyl transferase involved in the formation of lignin in maize) is capable of converting luteolin into chrysoeriol, as was suggested in sorghum, as a key step in the formation of tricin, a proposed initiator for the formation of lignin. So far, however, we have not been able to detect any formation of chrysoeriol in our yeast strains. Objective 3: Engineer maysin production in tomato(Year 2 - 3, Progress 15%):We analyzed two tomato genotypes (Castlemart and M82) for the accumulation of maysin precursors to decide which one would work best for expressing the maize pathway genes. As expected, tomato does not accumulate flavones in leaf or fruit tissues, and the analysis of the two flavanone precursors showed a better accumulation in M82. We then initiated the cloning of combinations of constitutive and trichome specific promoters with the maize flavone pathway enzymes that we have previously characterized. We also cloned the respective promoters upstream of a reporter gene and put them into transgenic plants to confirm that the expression of the promoters was as reported in the literature. Transgenic calli are currently growing and the process of tomato plant regeneration has been started.
Publications
|
|