Progress 06/01/13 to 12/14/14
Outputs
Target Audience: Scientific community (through a publication in Plant Physiology) Industry (through meetings with a corporate partner (Forage Genetics International) Government/Industry (through discussions with scientists and administrators from AgResearch/Grasslanz Technology, New Zealand). On the basis of the success we have made on this project, AgResearch/Grasslanz have entered into a new sponsored research agreement with the PI for work on engineering proanthocyanidins in row crops. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Dr Chenggang Liu (postdoctoral fellow) was appointed Research Assistant Professor at the University of North Texas. He worked on this project during the final year of no-cost extension, and gained experience in grant writing and personnel management in addition to his research training. How have the results been disseminated to communities of interest? Yes: To the scientific community (through a publication in Plant Physiology) To industry (through meetings with a corporate partner (Forage Genetics International) To government/Industry (through discussions with scientists and administrators from AgResearch/Grasslanz Technology, New Zealand). On the basis of the success we have made on this project, AgResearch/Grasslanz have entered into a new sponsored research agreement with the PI for work on engineering proanthocyanidins in row crops. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In the second no-cost extension, we have achieved the following: We published our results about MYB5 and MYB14 in Plant Physiology (Liu, et al, 2014). Our results demonstrated that MYB5 and MYB14 act synergistically to promote PA biosynthesis in Medicago. In collaboration with our commercial partner, Forage Genetics International, we generated alfalfa plants co-expressing various transcriptional factors regulating PA biosynthesis, including MtLAP1, MtMYB5, MtMYB14, and others (proprietary). We analyzed the PA contents and compositions of these alfalfa lines co-expressing various transcription factors. We have begun a detailed metabolic analysis of mutants of Medicago truncatula with loss of function of anthocyanidin reductase and leucoanthocyanidin reductase, Outcomes Based on our transactivation assays, we found that MYB5 and MYB14 can synergistically activate the promoters of the key structural genes of PA biosynthesis, ANR and LAR, in an MtTT8 and MtWD40-1 dependent manner. More importantly, we demonstrated that MYB5 and MYB14 can form a quaternary complex with MtTT8 and MtWD40-1 in contrast to the general concepts that MYB5 and MYB14 form separate ternary complex with MTTT8 and MtWD40-1. Our results reveal a novel mechanism of PA biosynthesis regulation. Based on these results, we hypothesized that co-expression of MYB5 and MYB14 could enhance the PA content of alfalfa leaves. Since we had difficulty in obtaining alfalfa expressing high level of MYB5 and MYB14 independently, presumably due to the fact that high levels of PA inhibit the regeneration of plants from somatic embryos during the tissue culture procedure, we first generated alfalfa lines expressing moderate levels of MYB5 or MYB14 transcripts, and then crossed the MYB5 and MYB14 expressing plants to obtain alfalfa lines co-expressing MYB5 and MYB14. Our results indicated that co-expression of MYB5 and MYB14 significantly increase PA content in alfalfa leaves as judged by the DMACA staining technique. In collaboration with our commercial partner, we also generated alfalfa co-expressing MYB14 and other transcription factors (proprietary). Our results indicated that co-expression of additional TFs can enhance the PA content of MYB14 expressing alfalfa, to levels that could be sufficient to prevent pasture bloat in livestock. However, we also observed that when the harvested alfalfa material was stored for prolonged times More than 2 months, the extractable soluble PA level dropped dramatically. We believe that the oxidation process after harvest is converting the soluble PA into insoluble PA, suggesting a need for additional studies to better understand the PA oxidation and polymerization process in alfalfa. The PA composition analyses of these improved alfalfa lines indicated that they produce exclusively epicatechin-based PA, similar to the PA produced in alfalfa seed coats. The mean degree of polymerization ranged from 2 to 15. In addition, we also observed that there are significant levels of anthocyanin in these PA producing alfalfa. We also observed that a MYB transcription factor, MtMYB2, which functions as an suppressor of anthocyanin biosynthesis, is strongly induced by MYB5 and the MYB transcription factor LAP1; this suggests that plants producing high levels of PAs may actively suppress anthocyanin biosynthesis to prevent anthocyanin toxicity. Genetic down-regulation of anthocyanin repressor expression in strongly PA producing alfalfa may therefore lead to increased PA production without accumulation of excess anthocyanin. Experiments to test this hypothesis are currently underway, using Medicago hairy roots as a model system. Analysis of mutants of Medicago truncatula with loss of function of anthocyanidin reductase (ANR) or leucoanthocyanidin reductase (LAR) has led us to new hypotheses about the pathway to PAs in M. truncatula and alfalfa. Essentially, loss of function of ANR leads to an expected phenotype, with reduction of PA level and accumulation of anthocyanins, a similar phenotype to that seen in Arabidopsis. However, the LAR loss of function mutant is impacted in PA biosynthesis, even though the PAs affected do not appear to contain catechin, the expected product of LAR. We believe that LAR has functions in PA biosynthesis beyond the formation of catechin. In summary, the work supported by this project has led to major breakthroughs in understanding the PA pathway, and in the engineering of proanthocyanidins for forage quality improvement in alfalfa.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Liu, C., Jun, J and Dixon, R.A. (2014). MYB5 and MYB14 play pivotal roles in seed coat polymer biosynthesis in Medicago truncatula. Plant Physiology 165, 1424-1439.
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Progress 06/01/13 to 05/31/14
Outputs
Target Audience: Please note that, although the request to transfer this grant from the Noble Foundation to the University of North Texas was made in early 2013, with a planned start date of June 1, 2013, the funds were not transferred and available to the PI until 3/20/2014 (approximately 2 months ago). This therefore impacts the progress in all criteria. However, we do have progress to report. Target audiences include: Commercial partners (Forage Genetics International, with whom we shared information in April 2014 and who are working with us towards commercialization of tannin-containing alfalfa, and AgResearch New Zealand, who we met with earlier in May 2014 and who have expressed an interest in funding tannin technology in other crops). American Society of Nutrition. Dr Dixon presented a lecture, entitled "Biosynthesis of flavanols within plants and bioavailability to humans"in a symposium on flavanols at a session of the ASN in San Diego in early May 2014. Changes/Problems: This project at UNT started 9 months late due to problems in transferring the funds (closing down the Noble Foundation grant and starting the new award at UNT). The problem was not at UNT's end. Work continued during this down-period as Dr Liu was hired and paid by UNT, but the funds for the operating expenses did not arrive at UNT until March 2014. What opportunities for training and professional development has the project provided? Dr Chenggang Liu, who is performing the research with salary funding from UNT, wrote the manuscriptdescribed above, with assistance for the PI. How have the results been disseminated to communities of interest? Through verbal communication (research presentations) to commercial partners What do you plan to do during the next reporting period to accomplish the goals? Complete the analysis of LAR and ANR mutants. Analyze transgenic alfalfa expressing both MYB14 and MYB5 transcription factors, Based on in vitro transactivation assays, we predict that these plants will accumulate large amounts of tannins, unless these should prove toxic
Impacts
What was accomplished under these goals?
1. Alfalfa plants expressing MYB5, MtPAR and Mt LAR have been supplied to our commerical partner Forage Genetics International and have entered their breeding program. Data indicate success in engineering tannins in the aerial portions of the plant. 2. Studies on the effects of MYB5 and other transcription factors on the transactivation of the ANR and LAR promoters have been completed. Data suggest the operation of a quaternary transcription complex. These results have been written up, and the manuscript below is currently in review with Plant Physiology: Chenggang Liu, Ji Hyung Jun and Richard A. Dixon. MtMYB5 and MtMYB14 play pivotal roles in seed coat polymer biosynthesis in Medicago truncatula 3. We have made the exciting discovery that mutants in Medicago LAR, believed to be simply an enzyme of catechin biosynthesis, completely lack soluble tannins (which are primarily made of epicatechin), but produce more insoluble tannins, whereas mutants in ANR (the enzyme that makes epicatechin) lack both soluble and insoluble tannins. This completely changes our picture of tannin biosynthesis and the roles of these enzymes. We are currently performing metabolomic analysis on the mutants.
Publications
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