Progress 03/01/15 to 02/28/19
Outputs
Target Audience:The target audience of this basic plant biology project is broad, from pharmaceutical and food industry that would like to improve the production of plant natural products (e.g. morphine and codeine, isoquinoline alkaloids, natural pigments derived from tyrosine) to agriculture sectors that are interested in improving crop nutritional values (e.g. betalain and vitamin E contents) and in enhancing crop resistance to abiotic and biotic stresses. In order to access the broad audience, the obtained findings were presented in academic meetings and are submitted for publishing in a peer-reviewed journal. We have also conducted an outreach program called "Pigment-Art" during the Science Nights at various elementary schools in the City of Madison during the entire duration of this project. In this event, children enjoy painting beautiful colors of betalains and other pigments, while parents and older children had opportunities to learn about the nutritional and pharmacological values of plant natural products. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities for postdoc, graduate and undergraduate students to be trained in the interdisciplinary field of basic plant biochemistry and crop science. Matthew Mirkes and Kathrina Wigg (hourly undergrads) from Goldman Lab and Samuel Lopez-Nieves (minority graduate student supported by fellowship), Minmin Wang and Marcos Oliveira (postdocs), and Sai Batchu and Daniel Griffith (undergrads for independent research) from Maeda Lab have been working together to generate different transgenic lines, grow different varieties of beets, and harvest them for various molecular, biochemical, and chemical analyses. Maeda lab members were also involved in the Pigment Art outreach activity to disseminate the importance of plant natural products and metabolism research in improving human nutrition and medicine. How have the results been disseminated to communities of interest?The majority of our findings have been already published in peer-reviewd journals (Wang et al., 2017, Lopez-Nieves et al., 2018, de Oliveira et al., 2018, Timoneda et al., 2018), which were further features in various publications, e.g. the Cover and Commentary of New Phytol., and, Nature Plants, USDA-NIFA News, New York Times and BBC (spanish). Additionally, we are still preparing at least two more manuscripts to be submitted soon (Lopez-Nieves et al., in preparation). We have also conducted an outreach program called "Pigment-Art" at the Science Nights events at various elementary schools in the City of Madison from 2015 to 2019. In this event, children enjoy painting beautiful colors of betalains and other pigments, while parents and older children had opportunities to learn about the nutritional and pharmacological values of plant natural products. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: We determined how L-tyrosine (Tyr) is synthesized in beets (B. vulgaris) and also uncovered evolutionary history of the Tyr and betalain biosynthetic pathways in Caryophyllales plants, to which B. vulgaris belongs. We discovered Tyr-synthesizing arogenate dehydrogenase (ADH) enzymes that are not feedback inhibited by Tyr in beets and other related species. Expression of the beet ADH enzyme and the relaxation of the regulation of Tyr biosynthesis led to increased accumulation of Tyr in Arabidopsis thaliana and Nicotiana benthamiana, further providing in vivo evidence for the critical role of the ADH feedback regulation in overall production of Tyr. Also, chemical analyses conducted in various beet cultivars suggested that different strategies will be needed to enhance the production of betalains in red and yellow beets: the supply and utilization of Tyr precursor are limited in red and yellow beets, respectively. The novel insights and enzymes obtained in this study can facilitate our breeding and engineering efforts to improve the production of these natural pigments as well as other Tyr-derived plant natural products, such as vitamin E and morphine alkaloids (Maeda, Lopez-Nieves, de Oliveira, 2018 patent). Key outcomes: Betalain pigments are unique to the plant order Caryophyllales and synthesized from the aromatic amino acid L-tyrosine (Tyr). We investigated the Tyr biosynthetic pathway in table beets (Beta vulgaris L.), which produce high levels of betalains. First, we found that, like most plants, B. vulgaris synthesizes Tyr via plastidic arogenate dehydrogenases (ADH), which were encoded by two ADH genes (BvADH1 and BvADH2, Lopez-Nieves et al., 2018 New Phytol.). However, unlike BvADH1 and other plant ADHs that are strongly inhibited by Tyr, BvADH2 exhibited relaxed sensitivity to Tyr, suggesting that the relaxed regulation of Tyr biosynthesis might have contributed to the production of Tyr-derived betalain pigments in table beets. However, no amino acid sequence difference was found in BvADH2 among red, yellow, sugar, white, and wild sea beets, suggesting that Tyr insensitivity of BvADH2 was not selected during domestication. Phylogenetic analysis of BvADH1 and BvADH2 orthologs, which were identified from transcriptome data of nearly two hundred Caryophyllales plants, showed that BvADH2 orthologs emerged before the appearance of betalain pigmentation and lost in Caryophyllaceae and Molluginaceae, two anthocyanins-producing families. Furthermore, Tyr-insensitive ADH2 orthologs were distributed among betalain-producing Caryophyllales. These results highlight critical contribution of the relaxation of Tyr pathway regulation to the evolution of betalain pigmentation (Lopez-Nieves et al., 2018 New Phytol.) While characterizing de-regulated ADH enzymes (i.e. BvADH2 orthologs) across the order Caryophyllales, we found that some Caryophyllales species have BvADH2 orthologs but do not produce betalains. This raised a question to why these plants still maintain feedback insensitive ADH enzymes. The GCMS-based metabolite profiling across the Caryophyllales phylogeny reveled that ADH2-containing species accumulate a large quantity of Tyr-derived natural products, besides betalain pigments, which include tyramine, L-DOPA, dopamine, and epinephrine derivatives. These results support that regulation of Tyr biosynthetic pathways play key roles in the production and evolution of plant natural products derived from Tyr (Lopez-Nieves, in preparation). To test if the identified Tyr-insensitive BvADH2 could actually function in vivo to enhance the production of Tyr, BvADH2 as well as BvADH1 and empty vector were introduced into a heterologous system, i.e. Arabidopsis thaliana, which only possesses Tyr-inhibited ADH enzymes. Significantly, transgenic lines expressing BvADH2, but not BvADH1 or empty vector, accumulated up to ~100-fold higher levels of tyrosine relative to controls. Thus, the Tyr-insensitive BvADH2 enzyme can be used to enhance production of Tyr (Oliveira et al., 2018). The results also indicate that the Tyr-mediated feedback regulation of the ADH-catalyzed reaction is the key regulatory step of Tyr biosynthesis in plants. In collaboration with Samuel Brockington's lab at University of Cambridge, we also demonstrated that expression of Tyr-insensitive BvADH2, together with the betalain biosynthetic enzymes, in Nicotiana benthamiana leads to significantly elevated production of the Tyr-derived natural products, betalains. To identify amino acid residues responsible for the Tyr insensitivity of Caryophyllales ADH2 enzymes, we conducted structure-function analysis by taking advantage of the large number of recently-duplicated BvADH2 and BvADH1 orthologs having distinct Tyr sensitivity. Their alignment identified 14 residues uniquely conserved among BvADH2 but not BvADH1 orthologs. Mapping these candidate residues onto a ADH2 protein structure, modeled based on the soybean PDH crystal structure, showed that only one residue Asp208 (Glu204 in ADH1) is at the active site. The D208E mutation indeed made ADH2 enzymes more sensitive to Tyr inhibition than wild-type. Furthermore, the reciprocal Glu to Asp mutation relaxed Tyr sensitivity of ADH1 enzymes. These results demonstrate that the replacement of the single acidic Asp/Glu residue plays a key role in altering the Tyr sensitivity of ADH enzymes of Caryophyllales (Lopez-Nieves, in preparation). The identied mutations will be useful tools to be introduced in other plants to de-regulate Tyr biosynthesis through gene-editing technology without using trangenic approaches especially in crops. To further identify possible limitation of betalain production in table beets, we conducted comparative analyses of betalains and their precursor, Tyr, in cultivars producing different kinds and levels of betalain pigments. Consistent with previous studies, red beets generally have >5-10 fold higher betalains than yellow beets. Interestingly, the levels of Tyr negatively correlated with those of betalains and were higher in yellow than red beets, suggesting that yellow beets are not efficiently utilizing tyrosine for pigment production. On the other hand, sugar and white beets, which accumulate very little betalain pigments, showed low levels of Tyr, suggesting that the supply of Tyr is reduced in beet cultivars producing high levels of sugars. Based on the observation, increased production of the Tyr precursor will be required to further increase betacyanin production in red beets, whereas better utilization of the accumulated Tyr can further improve betaxanthin production in yellow beets. Implication of the results: Our results addressed our first question of the project, "How do plants synthesize Tyr?" and uncovered critical contribution of the altered regulation of the Tyr pathway to the production of downstream plant natural products, i.e. betalain pigments and other Tyr-derived plant natural products. We also addressed our second question of "How can we enhance production of tyrosine-derived betalain pigments?". Clearly, different approaches, including de-regulation of the upstream Tyr pathway, are needed to improve the production of different types of betalains. The unique enzymes and their key amino acid residues identified from the order Caryophyllales will provide useful tools to boost the production of Tyr and Tyr-derived natural products, including betalains. Given that betalain pigments are the major natural red color dye produced in the US and the table beets are the major source, successful improvement of betalain contents in beets will help sustainable production of natural dyes and also enhance nutritional values of our food. Similar approaches can be also taken to achieve efficient production of other Tyr-derived plant natural products, such as vitamin E, morphine, and other alkaloids derived from Tyr.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Smith S.D., Angelovici R., Heyduk K., Maeda H.A., Moghe G.D., Pires J.C., Widhalm J.R., Wisecaver J.H. (2019). The Renaissance of Comparative Biochemistry. Am. J. Bot.. 106, 3-13. (non-corresponding authors are listed alphabeticvally)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Annual Meeting of the American Society of Plant Biologists (ASPB), Montreal July, 14-18, 2018 A Hidden Biochemical State that Facilitated the Evolution of Betalain Pigmentation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
1st Cologne Conference on Food for Future in Cologne, Germany September 7-9, 2018 Harnessing natural enzyme variants to build plant chemical platform
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Botany 2018, Evolution of Plant Chemical Diversity: Renaissance of Comparative Biochemistry in Rochester MN July 21-25, 2018 Evolutionary tug-of-war underlies betalain vs. anthocyanin pigmentations in Caryophyllales.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
de Oliveira M.V.V., Jin X., Chen X., Griffith D., Batchu S., Maeda H.A. (2018). Imbalance of tyrosine by modulating TyrA arogenate dehydrogenases impacts growth and development of Arabidopsis thaliana. Plant J. doi: 10.1111/tpj.14169
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Timoneda A., Sheehan H., Feng T., Lopez-Nieves S., Maeda H.A., Brockington S. (2018) Redirecting Primary Metabolism to Boost Production of Tyrosine-Derived Specialised Metabolites in Planta. Sci. Rep. 8; 17256
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Lopez-Nieves S., Pringle A., Maeda H.A. (2019) Biochemical characterization of TyrA dehydrogenases from Saccharomyces cerevisiae (Ascomycota) and Pleurotus ostreatus (Basidiomycota) Arch Biochem Biophys. doi: 10.1016/j.abb.2019.02.005. [Epub ahead of print]
|
Progress 03/01/17 to 02/28/18
Outputs
Target Audience:The target audience of this basic plant biology project is broad, from pharmaceutical industry that would like to improve the production of plant natural products (e.g. morphine and codeine, isoquinoline alkaloids derived from tyrosine) to agriculture sectors that are interested in improving crop nutritional values (e.g. betalain and vitamin E contents) and in enhancing crop resistance to abiotic and biotic stresses. In order to access the broad audience, the obtained findings were presented in academic meetings and are submitted for publishing in peer-reviewed journals. We have also conducted an outreach program called "Pigment-Art" during the Science Nights at Lake View Elementary School in the City of Madison on April 2017. In this event, children enjoy painting beautiful colors of betalains and other pigments, while parents and older children had opportunities to learn about the nutritional and pharmacological values of plant natural products. Changes/Problems:To complete the remaining experiments described above, no cost extension was requested for another one year, until Feb 29, 2019. What opportunities for training and professional development has the project provided?The project provided opportunities for postdoc, graduate and undergraduate students to be trained in the interdisciplinary field of basic plant biochemistry and crop science. During the third year, Samuel Lopez-Nieves (minority graduate student supported by fellowship), Minmin Wang and Marcos Oliveira (postdocs), and Daniel Griffith (undergrads for independent research) have been working together to generate different transgenic lines and grow different varieties of beets and harvesting for various molecular, biochemical, and chemical analyses. Samuel and Minmin's papers have been published, and Samuel also obtained the best oral presentation award at the SACNAS meeting. How have the results been disseminated to communities of interest?The major finding of this project has been published in New Phytologists this year and also on our campus news website, which attracted a broad interest beyond scientific community (e.g. NY Times, BBC). We are preparing additional manuscript to be submitted soon. We have also conducted an outreach program called "Pigment-Art" during the Science Nights at Lake View Elementary School in the City of Madison on April 2017. In this event, children enjoy painting beautiful colors of betalains and other pigments, while parents and older children had opportunities to learn about the nutritional and pharmacological values of plant natural products. What do you plan to do during the next reporting period to accomplish the goals?With the no-cost extension of this project, we will complete metabolite profiling of these BvADH2 expressing transgenic lines and some of beet cultivars to understand how altered activity of tyrosine biosynthesis will impact overall activity of aromatic amino acid pathways. Also, the structure-function analysis of the tyrosine-insensitive BvADH2 will be also continued, since the D208E ADH2 mutant is still partially insensitive to tyrosine inhibition as compared to BvADH1 orthologs, suggesting that yet an additional residue(s) must be involved in overall tyrosine sensitivity. To test the role of the remaining 13 non-active site residues, some or all of these residues will be simultaneously mutated in the D208E ADH2 mutant backgrounds. If time is allowed, the identified residues will be also introduced into other ADH enzymes (e.g tyrosine-inhibited Arabidopsis ADH) in vitro and in vivo to test their effects in a wider range of plant species.
Impacts
What was accomplished under these goals?
Impact: During the third year of the funding period, we have published our major finding of this project (Lopez-Nieves et al., 2017 New Phytologists), which attracted a wide range of interest and was featured in Nature Plants, the cover and commentary of New Phytologists, Spanish BBC, NIFA Update (https://content.govdelivery.com/accounts/USDANIFA/bulletins/1bc41a2), and New York Times (https://www.nytimes.com/2017/10/24/science/beets-red-enzymes.html). We found that beets and related plants became efficient in producing an amino acid tyrosine, which set the stage for their ancestors to develop the novel pigment pathway to make their characteristic betalain pigments. The novel insights and enzymes obtained in this study can facilitate our breeding and engineering efforts to improve the production of these natural pigments as well as other tyrosine-derived plant natural products, such as vitamin E and morphine alkaloids. Key outcomes: Betalain pigments are unique to the plant order Caryophyllales and synthesized from the aromatic amino acid L-tyrosine (Tyr). We investigated the Tyr biosynthetic pathway in table beets (Beta vulgaris L.), which produce high levels of betalains. In the previous year, comparative analyses of betalains and tyrosine in different beet cultivars revealed that the levels of tyrosine negatively correlated with those of betalains and were higher in yellow than red beets, suggesting that yellow beets are not efficiently utilizing tyrosine for pigment production (Wang et al., 2017). We also found that, like most plants, B. vulgaris synthesizes Tyr via plastidic arogenate dehydrogenases (ADH), which were encoded by two ADH genes (BvADH1 and BvADH2). However, unlike BvADH1 and other plant ADHs that are strongly inhibited by Tyr, BvADH2 exhibited relaxed sensitivity to Tyr. Phylogenetic analysis combined with recombinant enzyme characterization further revealed that Tyr-insensitive BvADH2 orthologs arose before betalain pigmentation in the Caryophyllales. These results highlight critical contribution of the relaxation of Tyr pathway regulation to the evolution of betalain pigmentation (Lopez-Nieves et al., 2017 New Phytologists). To further test the in vivo functionality of the identified Tyr-insensitive beet ADH enzyme, BvADH2 as well as BvADH1 and empty vector, as controls, were expressed in two heterologous systems, Arabidopsis thaliana and Nicotiana benthamiana, which only possesses Tyr-inhibited ADH enzymes. In both systems, expression of BvADH2, but not BvADH1 or empty vector, resulted in up to ~100-fold increased accumulation of tyrosine relative to controls. Thus, the Tyr-insensitive BvADH2 enzyme can de-regulate the tyrosine biosynthetic pathway and enhance the production of Tyr and possibly Tyr-derived natural products. Metabolite profiling of the BvADH2 expressing lines of A. thaliana and N. benthamiana also showed that the level of the other aromatic amino acid, phenylalanine, as well as its derivatives (e.g. sinapoyl malate) was reduced. Since tyrosine and phenylalanine biosynthesis shares the same upstream shikimate pathway and compete the arogenate substrate, these results suggest that de-regulation of tyrosine biosynthesis redirect carbon flow from phenylalanine to tyrosine biosynthesis in plants. While characterizing de-regulated ADH enzymes (i.e. BvADH2 orthologs) across the order Caryophyllales, we found that some Caryophyllales species have BvADH2 orthologs but do not produce betalains. This raised a question to why these plants still maintain feedback insensitive ADH enzymes. The GCMS-based metabolite profiling across the Caryophyllales phylogeny reveled that ADH2-containing species accumulate a large quantity of tyrosine-derived natural products, besides betalain pigments, which include tyramine, L-DOPA, dopamine, and epinephrine derivatives. These results further support that regulation of tyrosine biosynthetic pathways play key roles in the production and evolution of plant natural products derived from tyrosine. To identify amino acid residues responsible for the Tyr insensitivity of Caryophyllales ADH2 enzymes, we began structure-function analysis by taking advantage of the large number of recently-duplicated BvADH2 and BvADH1 orthologs having distinct Tyr sensitivity. Their alignment identified 14 residues uniquely conserved among BvADH2 but not BvADH1 orthologs. Mapping these candidate residues onto a ADH2 protein structure, modeled based on the soybean PDH crystal structure, showed that only one residue Asp208 (Glu204 in ADH1) is at the active site. The D208E mutation indeed made ADH2 enzymes more sensitive to Tyr inhibition than wild-type. Furthermore, the reciprocal Glu to Asp mutation relaxed Tyr sensitivity of ADH1 enzymes. These results demonstrate that the replacement of the single acidic Asp/Glu residue plays a key role in altering the Tyr sensitivity of ADH enzymes of Caryophyllales.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Lopez-Nieves S, Yang Y, Timoneda A, Wang M, Feng T, Smith SA, Brockington SF, Maeda HA (2018) Relaxation of tyrosine pathway regulation underlies the evolution of betalain pigmentation in Caryophyllales. New Phytol 217: 896908
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Schenck C.A. and Maeda H.A. Tyrosine Biosynthesis, Metabolism, and Catabolism in Plants. Phytochemistry
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Wang M., Lopez-Nieves S., Goldman I. and Maeda H.A.* (2017) Limited Tyrosine Utilization Explains Lower Betalain Contents in Yellow than Red Table Beet Genotypes. J Agric Food Chem. 65, 43054313.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Maeda H.A. (2017) Plant Metabolic Diversity: Interspecies Variations of Tyrosine Biosynthetic Pathway Architecture and Regulation. Fourth International Conference on Plant Metabolism (ICPM), Dalian, China July 16-20 (oral presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Maeda H.A. (2017) Plant Metabolic Diversity: Interspecies Variations of Tyrosine Biosynthetic Pathway Architecture and Regulation. Phytochemical Society of North America (PSNA), University of Missouri, MO August 5-9 (oral presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Lopez-Nieves S., and Maeda H.A. (2017). Deciphering the molecular mechanism underlying relaxed regulation of tyrosine biosynthesis in Caryophyllales. SACNAS National Conference, Salt Lake, UT October 19-21 (oral presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Lopez-Nieves S., Yang Y., Oliveira M., Feng T., Griffith D., Smith S.A., Brockington S.F., and Maeda H.A. (2017) Relaxed Regulation of Tyrosine Biosynthesis Underlies the Evolutionary Expansion of Diverse Plant Natural Products. Gordon Research Conference on Plant Metabolic Engineering, Waterville Valley, NH July 9-14, (poster presentation)
- Type:
Theses/Dissertations
Status:
Awaiting Publication
Year Published:
2017
Citation:
Lopez-Nieves S. (2017) The Tyrosine Biosynthetic Pathway and Its Regulation in the Plant Order Caryophyllales. Ph.D. thesis, University of Wisconsin-Madison, Department of Botany
|
Progress 03/01/16 to 02/28/17
Outputs
Target Audience:The target audience of this basic plant biology project is broad, from pharmaceutical industry that would like to improve the production of plant natural products (e.g. morphine and codeine, isoquinoline alkaloids derived from tyrosine) to agriculture sectors that are interested in improving crop nutritional values (e.g. betalain and vitamin E contents) and in enhancing crop resistance to abiotic and biotic stresses. In order to access the broad audience, the obtained findings were presented in academic meetings and are submitted for publishing in a peer-reviewed journal. We have also conducted an outreach program called "Pigment-Art" during the Science Nights at Huegel Elementary School in the City of Madison on April 2016. In this event, children enjoy painting beautiful colors of betalains and other pigments, while parents and older children had opportunities to learn about the nutritional and pharmacological values of plant natural products. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities for postdoc, graduate and undergraduate students to be trained in the interdisciplinary field of basic plant biochemistry and crop science. During the second year, Matthew Mirkes (hourly undergrad) from Goldman Lab and Samuel Lopez-Nieves (minority graduate student supported by fellowship), Minmin Wang and Marcos Oliveira (postdocs), and Sai Batchu and Daniel Griffith (undergrads for independent research) from Maeda Lab have been working together to generate different transgenic lines and grow different varieties of beets and harvesting for various molecular, biochemical, and chemical analyses. How have the results been disseminated to communities of interest?An initial part of the obtained result has been submitted for publication. The remaining results are in preparation to be submitted at the beginning of the year three. We have also conducted an outreach program called "Pigment-Art" during the Science Nights at Huegel Elementary School in the City of Madison on April 2016. In this event, children enjoy painting beautiful colors of betalains and other pigments, while parents and older children had opportunities to learn about the nutritional and pharmacological values of plant natural products. What do you plan to do during the next reporting period to accomplish the goals?Given that we have identified the key regulatory step of Tyr biosynthesis and some limiting factors for betalain synthesis in beets, we next plan to conduct following experiments. First, we will repeat metabolite profiling analyses from different beet cultivars but by growing them in the field to test if the same conclusions about the limiting factors can be drawn. We have tried growing beets in the field in the second year, but the heavy rain during the seedling stage washed away most of them. Given that the light intensity (and thus rate of photosynthesis) as well as other environmental factors are different between greenhouse and field, it is important to replicate the experiments. Also, expression data will be also obtained for betalain, tyrosine and shikimate pathway genes to correlate with the metabolite data. Second, we will conduct structure-function analysis of the Tyr-insensitive BvADH2 enzyme to identify critical amino acid residues responsible for this unique characteristic of BvADH2 Tyr-insensitivity. Comparison of amino acid sequences from over one hundred orthologs of Tyr-sensitive BvADH1 and Tyr-insensitive BvADH2 have already identified ~10 residues uniquely present among Tyr-sensitive orthologs or vise versa. These residues will be first mutated all together to test if we can convert Tyr-sensitive enzymes to Tyr-insensitive or vise versa. If so, we will narrow down key residues by converting them back individually step-by-step. The identified residues can be then used to introduce Tyr-insensitivity to a wide range of plant species to increase Tyr availability.
Impacts
What was accomplished under these goals?
Impact: During the second year of the funding period, we further demonstrated that relaxed regulation of L-tyrosine (Tyr) biosynthetic pathway leads to increased accumulation of Tyr in planta, further providing in vivo evidence for the critical role of the feedback regulation in overall production of Tyr and likely Tyr-derived natural products. Also, our new data suggest that different strategies will be needed to enhance the production of betalains in red and yellow beets: the supply and utilization of Tyr precursor are limited in red and yellow beets, respectively. Key outcomes: Betalain pigments are unique to the plant order Caryophyllales and synthesized from the aromatic amino acid L-tyrosine (Tyr). We investigated the Tyr biosynthetic pathway in table beets (Beta vulgaris L.), which produce high levels of betalains. From the first year we found that, like most plants, B. vulgaris synthesizes Tyr via plastidic arogenate dehydrogenases (ADH), which were encoded by two ADH genes (BvADH1 and BvADH2). However, unlike BvADH1 and other plant ADHs that are strongly inhibited by Tyr, BvADH2 exhibited relaxed sensitivity to Tyr. Phylogenetic analysis combined with recombinant enzyme characterization further revealed that Tyr-insensitive BvADH2 orthologs arose before betalain pigmentation in the Caryophyllales. These results highlight critical contribution of the relaxation of Tyr pathway regulation to the evolution of betalain pigmentation (Lopez-Nieves et al., submitted) To further test if the identified Tyr-insensitive BvADH2 could actually function in vivo to enhance the production of Tyr, BvADH2 as well as BvADH1 and empty vector were introduced into a heterologous system, i.e. Arabidopsis thaliana, which only pocesses Tyr-inhibited ADH enzymes. After confirming the single insertion events of these transgenes, multiple independent lines were analyzed for soluble metabolites including tyrosine. The results showed that transgenic lines expressing BvADH2, but not BvADH1 or empty vector, accumulated up to ~100-fold higher levels of tyrosine relative to controls. Thus, the Tyr-insensitive BvADH2 enzyme can be used to enhance production of Tyr and possibly Tyr-derived natural products. To further identify possible limitation of betalain production in table beets, we conducted comparative analyses of betalains and their precursor, tyrosine, in cultivars producing different kinds and levels of betalain pigments. Consistent with previous studies, red beets generally have >5-10 fold higher betalains than yellow beets. Interestingly, the levels of tyrosine negatively correlated with those of betalains and were higher in yellow than red beets, suggesting that yellow beets are not efficiently utilizing tyrosine for pigment production. On the other hand, sugar and white beets, which accumulate very little betalain pigments, showed low levels of tyrosine, suggesting that the supply of tyrosine is reduced in beet cultivars producing high levels of sugars. Based on the observation, increased production of the tyrosine precursor will be required to further increase betacyanin production in red beets, whereas better utilization of the accumulated tyrosine can further improve betaxanthin production in yellow beets. Implication of the results: Our results from the past two years have already addressed our first question of the project, "How do plants synthesize tyrosine?" and uncovered critical contribution of the altered regulation of the tyrosine pathway to the production of downstream plant natural products, i.e. betalain pigments. The results from the second year further addressed our second question of "How can we enhance production of tyrosine-derived betalain pigments?". Clearly, different approaches, including de-regulation of the upstream Tyr pathway, are needed to improve the production of different types of betalains. Given that betalain pigments are the major natural red color dye produced in the US and the table beets are the major source, successful improvement of betalain contents in beets will help sustainable production of natural dyes and also enhance nutritional values of our food.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Lopez-Nieves S., Yang Y., Smith S.A., Brockington S.F., and Maeda H.A. Relaxation of Tyrosine Pathway Regulation During the Evolution of Betalain Pigmentation in Caryophyllales. Submitted.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Maeda H.A. (2016) Relaxed Regulation of Tyrosine Biosynthesis During the Evolution of Betalain Pigmentation
Phytochemical Society of North America (PSNA), Davis, CA August 7th
(oral presentation)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Maeda H.A. (2015) Diversification of Tyrosine Biosynthetic Pathways in Plants Phytochemical Society of North America (PSNA), Urbana-Champaign, IL August 10th,
(oral presentation)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Lo?pez-Nieves S., Yang Y., Smith S.A., Brockington S.F., and Maeda H.A. (2016) Relaxation of Tyrosine Pathway Regulation Precedes the Evolution of Betalain Pigmentation in Caryophyllales. Annual meeting of the American Society of Plant Biologists, Austin, TX July
(oral presentation)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
L�pez-Nieves S. and Maeda H.A. (2016) Relaxation of Tyrosine Pathway Regulation Proceeds the Evolution of Betalain Pigmentation in Caryophyllales. Evolution Seminar, UW Madison March 15
(oral presentation)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Lo?pez-Nieves S., Yang Y., Smith S.A., Brockington S.F., and Maeda H.A. (2016) Relaxation of Tyrosine Pathway Regulation Precedes the Evolution of Betalain Pigmentation in Caryophyllales. SACNAS National Conference, Long Beach, CA October 13-15 (poster presentation)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Batchu S., Oliveira M. and Maeda H.M. (2016) Maximizing Tyrosine Accumulation in Arabidopsis thaliana. Symposium of Integrated Biological Sciences (IBS) Summer Research Program, UW-Madison, August 3rd (oral presentation)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2016
Citation:
Dewanjee A., L�pez-Nieves S., and Maeda H.A. (2016) Characterization of Arogenate Dehydrogenase Enzymes for Tyrosine Sensitivity. 17th annual Undergraduate Symposium, UW-Madison, April 14th (poster presentation)
|
Progress 03/01/15 to 02/29/16
Outputs
Target Audience:The target audience of this basic plant biology project is broad, from pharmaceutical industry that would like to improve the production of plant natural products (e.g. morphine and codeine, isoquinoline alkaloids derived from tyrosine) to agriculture sectors that are interested in improving crop nutritional values (e.g. betalain and vitamin E contents) and in enhancing crop resistance to abiotic and biotic stresses. In order to access the broad audience, the obtained findings were presented in academic meetings and are under preparation for publishing in a peer-reviewed journal. We have also conducted an outreach program called "Pigment-Art" during Saturday Science at Wisconsin Institute of Discovery on November 7th, 2015. In this event, children enjoy painting beautiful colors of betalains and other pigments, while parents and older children will have opportunities to learn about the nutritional and pharmacological values of plant natural products. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities for postdoc, graduate and undergraduate students to be trained in the interdisciplinary field of basic plant biochemistry and crop science. Samantha Milota (hourly undergrad) from Goldman Lab and Samuel Lopez-Nieves (minority graduate student supported by fellowship), Minmin Wang (postdoc), and Stephan Miller (undergrad for independent research) from Maeda Lab have been working together to grow different varieties of beets and harvesting for various molecular, biochemical, and chemical analyses. How have the results been disseminated to communities of interest?The obtained results are under preparation for publishing in a peer-reviewed journal. We have also conducted an outreach program called "Pigment-Art" during Saturday Science at Wisconsin Institute of Discovery on November 7th, 2015. In this event, children enjoy painting beautiful colors of betalains and other pigments, while parents and older children had opportunities to learn about the nutritional and pharmacological values of plant natural products. What do you plan to do during the next reporting period to accomplish the goals?Given that we have uncovered that relaxed regulation of the ADH enzyme contributed to the production of betalain pigments in beets and betalain-producing Caryophyllales, next we would like to examine if tyrosine synthesis is a limiting factor for betalain production in table beets. To address this question, we plan to conduct two major experiments: First is to analyze the levels of different metabolites (tyrosine, betalains, as well as tyramine and tocopherols, other tyrosine-derived natural products) across different beet cultivars and species of Caryophyllales. If the levels of tyrosine and other tyrosine-derived metabolites (e.g. tyramine and tocopherols) are low in betalain accumulated plants, it suggests that tyrosine production is limited and further increase in tyrosine production could lead to elevated betalain pigmentation. If the levels of tyrosine, tyramine and/or tocopherols are elevated in betalain accumulated plants, tyrosine production is unlikely to be limited and thus further improvement in betalain biosynthetic pathway will be a more promising approach. We have already set up various analytical procedures (e.g., GCMS for tyrosine, tyramine, HPLC for tocopherols and betalains) and harvested plant tissues to be analyzed in this coming year. Second approach is to introduce different types of tyrosine-insensitive ADH and PDH enzymes in beet roots using hairy root transformation. Previous studies from my and other labs have identified several ADH or PDH enzymes that are no longer inhibited by tyrosine, such as cyanobacteria Synechocystis ADH (Legrand et al., 2006), soybean and Medicago PDHs (Schenck et al., 2015), and Medicago ADH enzyme (Schenck et al., unpublished data). We will over express these enzymes as well as Arabidopsis tyrosine-sensitive ADH enzyme (as a negative control) in beet roots and examine its impact on the tyrosine/betalain pathways by similar metabolite analyses described above. We are planning to generate vector constructs and transgenic hair roots in this coming year and to conduct metabolite analyses during the final year.
Impacts
What was accomplished under these goals?
Impact: During the first year of the funding period, we were able to define how tyrosine is synthesized in beets (Beta vulgaris) and also uncovered evolutionary history of the tyrosine and betalain biosynthetic pathways in Caryophyllales plants, where B. vulgaris belongs. We identified one tyrosine pathway enzyme from beet that exhibited relaxed inhibition by tyrosine, providing a useful tool to improve the production of tyrosine-derived natural products. Key outcomes: To determine the tyrosine biosynthetic pathway that operates in beets, protein crude extracts were prepared from different parts of beet tissues and conducted enzyme assays for prephenate dehydrogenase (PDH) and arogenate dehydrogenase (ADH), two alternative enzymatic activities that determine tyrosine biosynthetic route. Both leaf and root tissues of beets exhibited ADH but not PDH activity, suggesting that beets synthesize tyrosine via the ADH pathway. Furthermore, we found that the beet genome contained two genes (BvADH1 and BvADH2) encoding enzymes having ADH activity. When green fluorescence protein (GFP)-fused BvADH proteins were expressed in Arabidopsis protoplasts and analyzed for their subcellular localizations, both BvADH1 and BvADH2 were targeted to the plastids, where aromatic amino acid biosynthesis usually takes place in plants. These biochemical and molecular data indicate that beets produce tyrosine via ADH pathway that is localized within the plastids. ADH (and PDH) enzymes are usually strongly feedback inhibited by tyrosine. Interestingly, however, BvADH2 (but not BvADH1) exhibited reduced sensitivity to Tyr inhibition, suggesting that the relaxed inhibition of tyrosine biosynthesis might have contributed to the production of tyrosine-derived betalain pigments. However, no nucleotide difference was found in BvADH2 among red, yellow, sugar, white, and wild sea beets, suggesting that Tyr insensitivity of BvADH2 was not selected during domestication. Phylogenetic analysis of BvADH1 and BvADH2 orthologs, which were identified from transcriptome data of nearly two hundred Caryophyllales plants, showed that BvADH2 orthologs emerged before the appearance of betalain pigmentation and lost in Caryophyllaceae and Molluginaceae, two anthocyanins-producing families. Furthermore, Tyr-insensitive ADH2 orthologs were distributed among only in betalain-producing Caryophyllales. These findings suggest that lineage-specific Tyr-insensitive ADH2 underlies the evolution of betalain pigmentation in Caryophyllales. Thus, our current model is that a Tyr-insensitive ADH2 enzyme emerged at the basal Caryophyllales, which led to increased accumulation tyrosine and likely provided a unique opportunity for this particular lineage of plants to produce betalain pigments derived from tyrosine. Implication of the results: Our results from the first year has already addressed our first question of the project, "How do plants synthesize tyrosine?" and uncovered critical contribution of the altered regulation of the tyrosine pathway to the production of downstream plant natural products, i.e. betalain pigments. These results also provided crucial information to address our second question of "How can we enhance production of tyrosine-derived betalain pigments?". Our data clearly demonstrated the critical role of the regulation of tyrosine biosynthesis in betalain production. Thus, we can employ different approaches to further increase tyrosine synthesis in beets for improved production of betalains. Given that betalain pigments are the major natural red color dye produced in the US and that the table beets are the major source, successful improvement of betalain contents in beets will help sustainable production of natural dyes and also enhance nutritional values of our food.
Publications
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