Progress 10/01/13 to 09/30/18
Outputs
Target Audience:The overall goal of this project is to make food items healthier for the consumer. Carotenoids are known to have health benefits so crops fortified with enhanced carotenoid levels would be benefical to consumers and would decrease the cost of health care in the US. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
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?
Nothing Reported
Impacts
What was accomplished under these goals?
Plants with enhanced carotenoid content in their seeds were successfully produced. It was determined that enhanced b-carotene levels also enhanced the overall protein content and seed size. All of these traits would be beneficial to the consumer.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Schmidt, MA, Pendarvis, K (2017) Proteome rebalancing in transgenic Camelina occurs within the enlarged proteome induced by B-carotene accumulation and storage protein suppression. Transgenic Research 26(2): 171-186
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Wing, R.A, M.D. Purugganan & Q. Zhang. The rice genome revolution: from an ancient grain to Green Super Rice. 2018. Nature Reviews Genetics [Article & Cover] 19:505-517.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Lv, Shuwei, W. Wu, M. Wang, R.S. Meyer, M.N. Ndjiondjop, L. Tan, H. Zhou, J. Zhang, Y. Fu, H. Cai, C. Sun, R.A. Wing, Z. Zhu. Genetic control of seed shattering during African rice domestication. 2018. Nature Plants, 4:331337.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Wang, W., R. Mauleon, Z. Hu, D. Chebotarov, S. Tai, Z. Wu, M. Li, T. Zheng, R.R. Fuentes, F. Zhang, L. Mansueto, D. Copetti, M. Sanciangco, K.C. Palis, J. Xu, C. Sun, H. Zhang, B. Fu, Yongming Gao1,5, Xiuqin Zhao1, Fei Shen9, Xiao Cui3, Hong Yu10, Zichao Li9, M. Chen, J. Detras, Y. Zhou, X. Zhang, Y. Zhao, D. Kudrna, C. Wang, R. Li, B. Jia, J. Lu, X. He, Z. Dong, J. Xu, Y. Li, M. Wang, J. Shi, J. Li, D. Zhang, S. Lee, W. Hu, A. Poliakov, I. Dubchak, V.J. Ulat, F.N. Borja, J.R. Mendoza, J. Ali, J. Li, M. Yang, Y. Niu, Z. Yue4, M.E.B. Naredo, J. Talag, X. Wang, J. Li, X. Fang, Y. Yin, J.C. Glaszmann, J. Zhang, J. Li, R.S. Hamilton, R.A. Wing*, C. Wei*, J. Ruan*, G. Zhang*, K.L. McNally*, N. Alexandrov*, Z. Li*, H. Leung (*Co-corresponding authors). 2018. Harnessing natural variation across 3,000 rice genomes: SNP discovery, population structure and genome diversity. 2018. Nature [Article] 557:43-49.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:This project focuses on the biofortification of oilseed crops with nutraceutical carotenoids, zeaxanthin or astaxanthin. Zeaxanthin enhanced seeds would benefitthe health of US consumers as a low consumption of this carotenoid in diets has been linked to the development of Age-related Macular Degeneration (AMD) -- the leading cause of irreversible vision loss in Americans age 55 years and older andcurrently estimated to affect 10 million Americans. The state of Arizona has a disproportionate elderly population likely due to its warm dry climate making it a retirement destination. In Arizona, the elderly population (over 60 yrs of age) will triple in size and represent 26% of the population in 2020. The audience for the second desired carotenoid would be the aquaculture industry as well as the general public. The carotenoid, astaxanthin, is used as a colorant in farm-raised salmon and today is currently supplied by chemical production resulting in costly inefficient processes that releaseharsh chemicals into the environment. Due to the difficultly of its chemical production, this compound can constitute up to 35% of the total production costs for the aquaculture industry. Making a renewable and sustainable source of this colorant in seeds would significantly benefit the global aquaculture community and the public by reducing chemical runoffs and reducing the costs of farm-raised fish. Changes/Problems:As Camelina is a fast-growing oilseed crop and easy to transform by floral dip, most constructs have been transformed into Camelina to show proof-of-concept before moving the genes into soybean. Soybean is a labor intensive, low transformation frequency (3%) process so it is difficult for students to complete during a term. What opportunities for training and professional development has the project provided?The project has been performed by an honor biochemistry student project for credit. The student has learned many molecular biology techniques including gene cloning, plasmid construction, plant transformation and DNA extraction/analysis. 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?The next step in the zeaxanthin and astaxanthin objectives is to analyze the transgenic Camelina seeds for their carotenoid content and composition using HPLC analysis and comparethe results to their counterpart nontransgenic seeds. If the seeds contain the desired carotenoids then we will further characterize the plants by showing their expression of the inserted genes and work towards a manuscript submission. The soybean lines that were produced overexpressing plastoglobulin will be crossed with the already produced b-carotene enhanced seeds to obtain double transgenicseeds. These seeds will then be analyzed for carotenoid content to determine if the plastoglobulin overproduction allowed for the accumulation of more b-carotene in the seeds.
Impacts
What was accomplished under these goals?
To producezeaxanthin in seeds we have cloned and produced a seed-specific expression cassette of a codon-optimized b-carotene hydroxylase and have introduced it into the oilseed crop Camelina along with the overexpression of phytoene synthase that had previously been shown to produce elevated levels of B-carotene in both soybean and Camelina seeds. Transgenic plants were obtained and initial screening of transgenic seeds shows that some seeds display theexpected yellow zeaxanthin color. To produce astaxanthin in seeds, we have cloned and produced codon-optimized b-carotene ketolase genes from two high accumulating astaxanthin species. We placed each ketolase gene under a strong seed-specific promoter along with a seed expressed phytoene synthase cassette. The combination of a ketolase and phytoene synthase should produce the desired astaxanthin. The goal is to determine which of the two ketolase enzymes has thehighestactivity by the production of the most astaxanthin within the seeds. We have produced transgenic Camelina seeds that should contain individually the two ketolase genes. The transgenic seeds have been harvested and appear to be a dark orange color, compared to the nontransgenic light brown color. We have cloned the soybean plastoglobulin gene and placed it under a strong seed specific expression cassette. We have produced 2 lines of transgenic soybeans containing the overexpression of the plastoglobulin gene. Seeds appear normal in color.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Due to a combination of an increase in life expectancy and aging & Baby Boomers, there will be 71 million Americans over the age of 65 in 2030 compared to 12 million in 1990. The state of Arizona (AZ) has a disproportionate elderly population likely due to its warm dry climate making it a retirement destination. In AZ, the elderly population (over 60 yrs of age) will triple in size and represent 26% of the population in 2020. Currently the leading cause of severe vision loss in Americans age 60 + is Age-related Macular Degeneration (AMD). The photoreceptors of the retina are the neurons that respond to light and are responsible for vision. It is the loss of photoreceptors that causes blindness from AMD. Studies on humans have demonstrated the beneficial effects of lutein and zeaxanthin in preventing the onset of AMD or its progression. There are no adverse effects to ingesting too much lutein or zeaxanthin, aside from a possible side effect of yellow discoloration of skin which is benign and reversible. It has been estimated that a 6-10 mg daily intake of zeaxanthin and lutein in Americans would have a $2.5 billion net savings to Medicare system over a 5 yr period. The current levels of these carotenoids, especially zeaxanthin, are too low to be beneficial in the typical American diet. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has been performed by a visiting scientist for 4 months andindependent studyundergraduate students. This project gives them the opportunity to learn about plant genomics, gene cloning, vector construction and plant transformation skills. 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?The putative transgenic plants will be grown in the next generation to generate enough seeds for analysis. Initially the seeds will be bulked and extracted for polar compounds. This extract will be run on thin layer chromatography for visual inspection of the production and accumulation of novel carotenoids in the seeds compared to the counterpart nontransgenic seeds. Seeds that are positive for TLC readings will be advanced to HPLC analysis for a more detailed and quantitative analysis of thecarotenoid content and composition of the transgenic seeds.
Impacts
What was accomplished under these goals?
We have made gene expression cassettes that will seed-specifically over-express two enzymes simultaneously.The first construct consists ofthe genes encoding fortwo enzymes: the phytoene synthase and the b-carotene ketolase. We produced two such expression cassettes using different ketolase enzymes from two different algae species that have been previously shown to vary in their catalytic function. We have then moved these cassettes into Agrobacterium for floral dip transformation of the model oil seed crop Camelina sativa. Primary transformant seeds have been obtained and are growing to collect seeds for analysis in the next generation. Secondly we have constructed gene expression cassettes with the seed-specific expression of both the phytoene synthase gene and the b-carotene hydroylase gene. These cassettes were moved into a binary vector and placed into Agrobacterium for floral dip in Camelina sativa plants. The initial putative transformant seeds have been obtained and plants are growing to produce enough seeds for analysis
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Due to a combination of an increase in life expectancy and aging 'Baby Boomers', there will be 71 million Americans over the age of 65 in 2030 compared to 12 million in 1990. The state of Arizona (AZ) has a disproportionate elderly population likely due to its warm dry climate making it a retirement destination. In AZ, the elderly population (over 60 yrs of age) will triple in size and represent 26% of the population in 2020. Currently the leading cause of severe vision loss in Americans age 60 + is Age-related Macular Degeneration (AMD). The photoreceptors of the retina are the neurons that respond to light and are responsible for vision. It is the loss of photoreceptors that causes blindness from AMD. Studies on humans have demonstrated the benefical effects of lutein and zeaxanthin in preventing the onset of AMD or its progression. There are no adverse effects to ingesting too much lutein or zeaxanthin, aside from a possible side effect of yellow discoloration of skin which is benign and reversible. It has been estimated that a 6-10 mg daily intake of zeaxanthin and lutein in Americans would have a $2.5 billion net savings to Medicare system over a 5 yr period. The current levels of these carotenoids, especially zeaxanthin, are too low to be beneficial in the typical American diet. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This research project has been the aim of two undergraduate student performing for credit independent research projects in my laboratory How have the results been disseminated to communities of interest?AMD is the result of damage to the macula that is located at the center of the retina in the eye. The macular damage results in central vision loss and can progress to complete blindness. Central vision loss makes every day activities, such as recognizing family and friends, cooking, driving, eventually impossible. Fortifying food items with zeaxanthin would greatly reduce the onset of this eye aliment in our elderly population resulting in a healthy active independent elderly lifestyle in turn lowering the cost of assisted living communities and medical services. This zeaxanthin biofortification project is akin to the addition of fluoride in the water to prevent tooth decay or iodine in salt to prevent iodine deficiency causing intellectual and developmental disabilities. AMD is preventable and zeaxanthin fortification is a means to alleviate this avoidable vision loss through good nutrition. What do you plan to do during the next reporting period to accomplish the goals?continue to grow the hydroyxlase transgenic soybean plants cross these plants with b-carotene accumulating plants to produce a double transgenic assess carotenoid content and composition in resultant seeds of above mentioned plants
Impacts
What was accomplished under these goals?
To address the low levels of the eye-healthy carotenoid zeaxanthin in American diets we are fortifying a common food item, soybean seeds, with the ability to both produce and accumulate biologically relevant levels of zeaxanthin. As zeaxanthin plays a role in photosynthesis in plants, it is a matter of engineering the endogenous pathway to over produce this desired carotenoid. Our previous research has successfully over-produced the carotenoid B-carotene to levels biologically relevant to prevent Vitamin A deficient - another eye aliment most prevalent in young children under the age of 5 in developing nations. The conversion of b-carotene to zeaxanthin involves a single enzymatic reaction. Progress to date includes cloning the necessary gene encoding for the desired enzyme, b-carotene hydroxylase, and re-introducing it under very high expression in transgenic soybean plants. The initial transgenic plants have been produced, confirmed to contain the inserted hydroxylase gene expression cassette and now are being grown to homozygousity for future carotenoid analysis. The hydroxylase plants will also be crossed with the enhanced b-carotene plants with the likelihood that the over accumulated b-carotene will be processed to zeaxanthin.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Schmidt MA, Parrott, WA, Hildebrand, DF, Berg RH, Cooksey A, Pendarvis K, He Y, McCarthy F and Herman EM (2015) Transgenic soybean seeds accumulating ?-carotene exhibit the collateral enhancements of high oleate and high protein content traits. Plant Biotechnology Journal 13: 590-600.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training activities include an undergraduate Honors Biochemistry student, Thomas Foss, conduced the experiments as an independent project. He learned to perform sterile tissue culture technIques, soybean biolistic transformation, DNA extraction, gene cloning techniques and polymerase chain reaction todetect inserted DNA in a plant's genome. He has recently graduated with this undergraduate degree and is seeking job opportunities in both the academic and industry sectorsin the soybean plant biotechnology field as a continuation ofthis project. How have the results been disseminated to communities of interest? This project of biofortifying soybean seeds with nutraceutical carotenoids has been displayed as part of an outreach committee booth. The event is called Plant Science Night and attracts hundreds of families of elementary aged school children. A display about the health benefits of carotenoids and a hands-on activity of thin layer chromatography to extract and detect carotenoids in common food items (fruits and vegetables) was given by this laboratory. What do you plan to do during the next reporting period to accomplish the goals? On-going: We will continue to fully regenerate the b-carotene hydroylase transgenic soybean lines produced to seed set. Seeds will be analyzed for lipid extraction and thin layer chromatography using commercially available zeaxanthin as a control. If samples are positive, high pressure liquid chromatography will be performed to quantitate the levels of enhanced zeaxanthin. In parallel, we will perform another set of soybean transformation experiments to produce at least a few more b-carotene hydroxylase lines. Eventually, the hydroxylase lines that display increased levels of zeaxanthin will be crossed with the already in hand b-carotene enhanced lines and the resultant seeds analyzed for both b-carotene and zeaxanthin.
Impacts
What was accomplished under these goals?
Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in Americans age 55 years and older. It currently affects 10 million Americans. It is estimated that a 6-10 mg daily intake of zeaxanthin and lutein in Americans would have a $2.5 billion net savings to Medicare system over a 5 yr period. Zeaxanthin is found in photosynthetic tissues as it protects against photooxidation. In foodstuff its concentration is measured in mg and it needs to be ingested at ~10mg / day to be beneficial to eye health - a magnitude of 100-1,000 fold too low. Objective: To over produce the nutraceutical carotenoid zeaxanthin in soybean seed tissue. Preliminary Results: Zeaxanthin is biosynthetically produced from b-carotene hydroxylase acting on b-carotene. We have already produced, and recently published an article on the enhancement of b-carotene in soybean seeds by seed-specifically expressing a bacterial phytoene synthase gene. We have the highest levels of carotenoid enhancement, nearly 1,500-fold increase, in any transgenic system with a single gene insertion. We will build on this accomplishment by adding the overexpression of b-carotene hydroxylase allowing for the conversion of b-carotene to zeaxanthin in soybean seeds. As soybean is a global commodity crop and the topmost US exported crop, its genome has been sequenced and readily available. By using the sequences of b-carotene hydroxylase proteins known from other plant species we identified the locus in soybean that likely encodes for that protein: Glyma13g39150. The gene has no introns and is 1482 nucleotides and encodes for a 493 amino acid protein. We designed primers to flank both the 5' and 3' open reading frame of the locus Glyma 13g39150 and added restriction sites NOT1 to each end for ease of cloning into already constructed seed-specific cloning vectors. Genomic DNA isolated from soybean leaf tissue was used as a template for PCR to amplify the gene of interest. We cloned the PCR amplicon into a TOPO vector and had the open reading frame sequenced. The sequence of the amplicon matched the locus Glyma 13g39150. With a NOT1 digest we moved this locus between seed-specific regulatory regions (glycinin promoter and terminator) and performed both digests and PCR to ensure the open reading frame was in the correct orientation. The plasmid was grown in E.coli and a large scale plasmid isolation performed. An undergraduate student doing an independent project was given the task of producing transgenic soybeans with this putative b-carotene hydroxylase seed-specific gene expression cassette. After inducing embryos, performing biolistic experiments, the cultures were maintained in hygromycin liquid selection for 6- 8 wks. A total of 8 putative transgenic hygromycin resistant embryo lines were isolated and tissue used as a template in genomic PCR. Five of the lines were deemed to be positive for the selectable hygromycin marker gene. Lines that were determined to be positive were then moved to maturation media to initiate regeneration to a plant. For each embryonic line, two sets of maturation experiments were conducted. For the first set, a few, still in sterile tissue culture, plantlets are emerging and the other set is still in the maturation phase. Typically we see the carotenoid color in the cotyledonary tissue of samples enhanced with seed-specific carotenoids but these culture did not deem any particularly noticeable color. The reasons for the lack of color in the cotyledonary-stage embyros might be (1) The hydroxylase protein alone may not produce a noticeable enhanced level of zeaxanthin (2) zeaxanthin is yellow in color and nontransgenic embyros are typically a green-yellow color so the enhancement of zeaxanthin may not be detectable visually (3) the introduced hydroxylase gene cassette may not be expressed in the lines produced.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Plant Bioteh J 2014 doi10.1111/pbi.12286
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