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Comparative analysis of tocopherol biosynthesis genes and its transcriptional regulation in soybean seeds. Vinutha T, Navita Bansal, Khushboo Kumari, Rama Prashat G, Rohini Sreevathsa, Veda Krishnan, Sweta Kumari, Anil Dahuja, S. K. Lal, Archana Sachdev, and Shelly Praveen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03448 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 2017
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Comparative analysis of tocopherol biosynthesis genes and its transcriptional regulation in
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soybean seeds.
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Vinutha T1, Navita Bansal1, Khushboo Kumari1, Rama Prashat G2, Rohini Sreevathsa3, Veda Krishnan1, Sweta Kumari1, Anil Dahuja1, S.K. Lal2, Archana Sachdev1 and Shelly Praveen1*
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1
Division of Biochemistry, IARI, New Delhi-110012
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Division of Genetics, IARI, New Delhi-110012
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3
National Research Centre on Plant Biotechnology
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* Corresponding author:
[email protected] 9
Abstract: Tocopherols composed of four isoforms (α, β, γ and δ) and its biosynthesis comprises
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of three pathways; Methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core
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pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at
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transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean
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genotypes was carried out, the results showed significantly differential expression of 5 genes; 1-
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deoxy-D-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) – from
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MEP; arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) – from SK and γ-
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tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were
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further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation
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network and gene clustering approaches, the results showed least and strong association of γ-
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TMT3/ tocopherol cyclase (TC) and DXR/DXS respectively with gene clusters of tocopherol
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biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation
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and intricacy of DXR/DXS genes in coordinating precursor pathways towards tocopherol
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biosynthesis in soybean seeds. Thus the present study provides insight into the major role of
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these genes regulating the tocopherol synthesis in soybean seeds.
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Key words: VitaminE, tocopherols, MEP, SK, soybean, phytyl 2P, Homogentisate, α-tocopherol
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Introduction:
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The unique nutritional profile [protein (40%) and oil (21%)] of soybean is a major reason for its
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cultivation and consumption in varied forms -- tofu, natto, miso, soy sauce, soy flour, soy protein
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or defatted soy meal and soymilk1,2. Besides, soybean also contains a number of health
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promoting compounds - isoflavones, anthocyanins, saponins and vitamin E (vit-E)
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Tocopherols are commonly termed as vit-E possesses potent antioxidant properties and are
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synthesized only by photosynthetic organisms i.e. higher plants and green algae. Because of the
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potent free radical scavenging properties, α-tocopherol is considered as the most powerful
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antioxidants 8. In plants, tocopherols are involved in intracellular signaling, stabilization of cell
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membrane, help in improving the oil and protein quality 9. In humans, intake of vit-E leads to
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decreased risk for cardiovascular disease, cancer, and caducity, aids in immune function, and
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prevents many degenerative diseases 10-12. In addition to enhancing the nutritional value of crops
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and human health benefits, increased levels of vit-E compounds have tremendous potential to
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improve the shelf life of seeds, seed vigor and seed oil quality including plant performance under
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stressful conditions that are linked to reactive oxygen species 13-15.
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Tocopherols are synthesized from condensation of two precurssor compounds; a polar
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chromanol group – homogentisate (HGA) synthesized through cytosolic shikimate (SK) pathway
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and an isoprenoid phytyl chain – phytyl diphosphate (PDP) synthesized from the plastidial
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methyl erythritol phosphate (MEP) pathway
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pathways of tocopherol-core pathway composed of twenty five biochemical steps-which
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provides metabolic influx into tocopherol-core pathway (Figure 1). These pathways (MEP and
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SK Pathway) not only act as precursor pathways for tocopherol biosynthesis, but also are
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connected to number of other metabolic pathways which include carotenoids, chlorophylls,
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gibberellins, phylloquinone and plastoquinone
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.
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3-7
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MEP and SK pathways are the upstream
. Tocopherols have four isoforms - α - , β-, γ-,
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and δ- tocopherol, of which α-tocopherol (α-toc) is considered as the most important form of vit-
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E for human health as it has ten-fold higher antioxidant activity than the other tocopherols and
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also been regarded as the most powerful protectant of cells-cellular components 18-20. In spite of
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all these benefits, α-toc proportion is only 0.50 by using edge –weighted force directed layout
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incorporated to the Cytoscape software
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performed according to Zhang and Horvath (2005).
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Results and Discussion
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Variation in tocopherol composition in soybean germplasm collections
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Duplicate soybean seed accession samples were grown and harvested from field experimental
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plots (ICAR-IARI facilities), collected and analyzed for tocopherol composition and content.
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Over 150 soybean germplasms were analyzed for tocopherol composition (Table S2). T-toc
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concentration was ranged from 29.72 µg/g (DS74) to 178.91 µg/g (Bragg). The predominant
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antioxidant in soybean seeds was γ-toc followed by δ and α-toc (Figure 2A). β+γ, δ, α-toc
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to analyze the expression patterns of
. The network was constructed with the genes presenting node
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. A signed network with threshold power (b) = 6 was
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content of 150 germplasm accessions averaged from 75.89±4.2, 22.11±1.65, 3.33±0.81 µg/g of
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seeds respectively. Except α-toc, significant (p
