Progress 07/01/08 to 06/30/12
Outputs
OUTPUTS: The overall goal of this project was to identify the genes and proteins that are the betalain secondary pigment pathway. This pathway supplants the anthocyanin pigment pathway in certain families in a single order of flowering plants, the Caryophyllales. Species that produce betalains and not anthocyanins include many minor but very important crop plants including: beets, Amaranthus, Quinoa, spinach, and cactus. Red betalain pigments from beet are the most common commercial natural food coloring. The pathway is fairly simple and the synthesis of the betalain ring structure is proposed to require three enzyme-mediated steps. Tyrosine is converted to LDOPA, one molecule of LDOPA is cleaved and recyclizes to form a yellow fluorescent compound, betalamic acid. Another molecule of LDOPA is further oxidized to cycloDOPA. Betalamic acid and cycloDOPA spontaneously condense to form red betacyanins. Betalamic acid will also condense with other amine groups, amino acids, etc to form yellow betaxanthins. When the project started, there was a single step known, a LDOPA dioxygenase, DODA, cleaves LDOPA which recyclizes to form betalamic acid. Our approach was to mine next generation sequence to identify genes encoding enzymes that we hypothesized would perform the steps leading to LDOPA from tyrosine, to cycloDOPA from LDOPA. These would then be tested using mutant complementation and reverse genetics approaches. We produced large databases of nextgen sequence for beet, amaranthus, and several other betalain species. We have developed methods for complementation and gene silencing in beet, Mirabilis, Celosia, and Amaranthus. The results have been disseminated in a variety of ways. The PI has given seminars, talks and posters at several meetings, Plant and Animal Genome Conference, American Society of Plant Biologist meetings, USDA PI meetings, etc. His students have presented posters at ASPB and departmental retreat meetings. The nextgen databases have been given to several researchers, most notably Dr. Mitch McGrath at Michigan State. The betalain genes and sequences discovered have been disseminated to Dr. McGrath, Dr. Friedrich Meischer, German beet breeder, Dr. Peter Felk, Cactus breeder at DArrigo Bros., and others. The project has resulted in a single very high profile publication in Nature Genetics and another has been positively reviewed and is in revision. PARTICIPANTS: Alan Lloyd. PI. Initiated the project and hypotheses. Developed the VIGS and complementation techniques. Analyzed nextgen data to idenfity potential betalain genes. Greg Hatlestad. Grad student who received his Ph.D. during the project. He performed much of the work on identification of R and Y and nextgen sequencing analysis. Rasika Sunnediniya. Current Grad student (currently out on maternity leave). Will complete her Ph.D. in next two years working on the betalain biosynthetic genes. Neda Akhavan. Current Grad student. Will complete her Ph.D. in next two years working on the betalain MYB regulatory locus. Lee Elam. Undergrad student who worked on cloning regulatory MYB into various plant and yeast vectors. Scott Cargyle. Undergrad student who worked on mapping of the Y locus. J. Mitch McGrath. USDA beet breeder and Michigan State faculty. Provided Beet Recombinant Inbred Line population, Beet BAC library, beet expertise. Irwin Goldman. U. Wisconsin faculty and beet breeder. Provided red beet cultivars, yellow/red segregating populations, beet expertise. TARGET AUDIENCES: The project provided valuable training opportunities for Grad students and Undergrads. Research opportunities included plant manipulations, molecular biology using plants, yeast and E. coli, nextgen sequencing and bioinformatics, HPLC and mass spec of small molecules, etc. All were required to participate in group meetings where they presented their research projects in a formal setting similar to meeting talks. They presented posters and talks at meetings. All members of the group participated in Science Fair activities at Cunningham Elementary School in the Austin Independent School District. Cunningham is a title 1 school with 65 % of students on free lunch, majority hispanic population and approximately 10% vietnamese population. The grad students produced a prefair workshop for students where they instructed students and families in the scientific method and brainstormed about project ideas and protocols. They then returned to the school to judge the science fair and interviewed the young scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The project resulted in the identification and characterization of three major steps in the betalain pathway. The nextgen sequence databases were mined for genes/enzymes thought likely to convert tyrosine to LDOPA, and LDOPA to cycloDOPA. Both steps have now been identified, the second step is published and it encodes a Cytochrome P450, CYP76AD1. This gene was silenced using Virus Induced Gene Silencing, developed for beet by this project, and silenced tissues produced yellow pigment. This indicated that this P450 performs the LDOPA to cycloDOPA step. The gene was then shown to complement mutant yellow beet to red. An insertional mutation in this gene was located in a yellow beet strain. And finally, the gene was mapped and shown to encode the R locus. The R locus was first identified by USDA geneticist, Wesley Keller in 1936. A gene/ enzyme the performs the tyrosine to LDOPA step has also now been identified. This is not quite ready for publication yet. The project resulted in the identification of a MYB gene that positively regulates the three enzymatic steps described above. This MYB was co-opted from the anthocyanin pathway, confirming one of our primary hypotheses as to how and why the betalain pigments are produced exactly like anthocyanins. This myb was mapped to the historic Y locus also identified by Keller in 1936. This work in under revision. As part of the project, nextgene sequencing was performed on flower tissue of Mirabilis jalapa, 4 Oclock flowers. Analysis of this sequence revealed that 26% of the reads were from a novel Carlavirus that infects Mirabilis. This work was published in 2011.
Publications
- Hatlestad G, Akhavan N, Sunnedeniya R, Elam L, Cargile S, Hembd A, Gonzalez A, McGrath M, Lloyd A (2013-in revision) A co-opted anthocyanin MYB regulates betalains: the historic beet Y locus is required to make beets red.
- Hatlestad G, Elam L, Gonzalez A, Lloyd A (2011) Mirabilis jalapa Latent Virus: a new Carlavirus infecting Four O clocks. Archives of Virology 156(11):2109-11.
- Hatlestad G*, Sunnedeniya R*, Akhavan N, Gonzalez A, Goldman I, McGrath M, Lloyd A (2012) The beet R locus encodes a new cytochrome P450 required for red betalain production. *co-first authors. Nature Genetics 44, 816-820 doi:10.1038/ng.2297
- Hatlestad G, Lloyd A (2013-in press) The betalain secondary metabolic network. Book Chapter.
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Progress 07/01/08 to 06/30/09
Outputs
OUTPUTS: Activities: We constructed a full-length cDNA library from Table beet cv. W357B hypocotyls expressing high amounts of betalain pigments. This is a Gateway technology library and it was recombined, en masse, into a yeast expression vector. The library is now being functionally screened in yeast for genes that confer betalain pigmentation in yeast. We constructed and performed 454 next generation sequencing on cDNA libraries from Table beet cv. W357B hypocotyls, and Amaranthus cruentus cv. Kerala Red hypocotyls. This sequencing provided similar results for each species: approximately 55,000 sequences of 330 bp average length, these sequences formed more than 6,500 contiguous sequences or putative genes for each. The contigs were annotated and then analyzed for genes known to be in the betalain pathway and for genes we hypothesized might be. Only 2 genes have been confidently placed in the pathway, a UGT and a DOPA dioxygenase (DODA). We did not find the UGT. However, in both species we found 2 DODA genes, 1 expressed at high levels and the other at much lower levels. The highly expressed, common DODA was the 19th highest expressed gene in Beet and 163rd highest in Amaranthus (out of more than 6,500 genes in each). These 2 data bases were BLASTED with genes that we hypothesized would be able to perform the other steps in the proposed betalain pathway. We identified a cytochrome P450 in each species that is expressed at approximately the same level as the common DODA (25th highest in beet and 200th highest in Amaranthus). We hypothesized that it might function as a monooxygenase on tyrosine, the first step in the pathway. We cloned and expressed the beet P450 in yeast. Alone, it does not seem to have any effect, however, when coexpressed with the DODA gene, we get yeast expressing yellow betalains. So we have discovered and cloned the first step in the pathway. This gene is now going into Arabidopsis alone and coexpressed with the DODA. One graduate, Rasika Sunnedeniys, has worked full time, two, Greg Hatlestad and Sarah Conte, worked half time and another full time grad student, Neda Akhavan, just joined the lab in August. In addition, one undergraduate, Kim Hosein, is working on the project. This project is providing valuable molecular and genetic training as well as critical thinking and problem solving skills. Products: New gene in the betalain pathway. New full-length Table beet cDNA library. New cDNA sequence database for Table beet. New cDNA sequence database for Amaranthus cruentus. Dissemination: We have provided the beet sequence database to a new collaborator, Dr. Mitch McGrath. Dr. McGrath is USDA sugarbeet breeder and he has supplied the Lloyd lab with a sugarbeet BAC library and sugarbeet x table beet Recombinant Inbred Line germplasm. We intend to collaborate on mapping the betalain genes in beets. We have provided the results of our recent P450 discovery to a new collaborator, Dr. Peter Felker. Dr. Felker is a cactus breeder at D'Arrigo Bros. Co. in Salinas, CA. Dr. Felker has supplied us with cactus germplasm and cactus fruit tissue expressing large amounts of betalains. We intend to attempt to clone the betalain gene cDNAs. PARTICIPANTS: PI: Alan Lloyd- organized and supervised work of others, grew plants, harvested tissue, generally helped grad students with various aspects of lab work, contacted collaborators. Grad Student: Rasika Sunnedeniya- performed gene cloning, sequencing, yeast transformation, plant transformation, tissue culture. Greg Hatlestad- performed bioinformatic analysis of 454 libraries, recombined Gateway beet library into yeast vector. Sarah Conte- performed cloning and sequencing, peformed confocal and other microscopic analysis. Undergraduate student: Kimberly Hosein- helped with general lab maintenance, planting, watering, washing, helped with plant transformations and other duties as requested by grad students. Collaborators: Mitch McGrath- USDA sugarbeet breeder, supplied sugarbeet BAC library, sugarbeet X table beet population germplasm, collaborating on mapping betalain genes in the beet genome. Peter Felker- D'arrigo Bros. Co., supplied cactus germplasm and tissue for attempts to clone betalain genes from cactus. Training: Students are receiving training in molecular biology, genetics, plant secondary metabolism. All students must present their findings to the lab group monthly. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: We have made the fundamental discovery of a new gene/enzyme in the betalain pathway. This is significant in and of itself, but even more so because the gene/enzyme, a cytochrome P450, is not what has been predicted in the literature for many years, i.e. a polyphenol oxydase or a laccase of some sort. We have also shown that this gene can be functionally expressed in heterologous species, yeast (other plants are being tested). Along with our novel finding that the DODA gene can be functionally expressed in yeast and heterologous plants, we are working towards building the entire betalain pathway in non-betalain producing species.
Publications
- No publications reported this period
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