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Molecular identification and functional characterization of a cyanogenic glucosyltransferase from flax (Linum unsitatissimum).

ABSTRACT: Flax seed has become consumers' choice for not only polyunsaturated alpha-linolenic fatty acid but also nutraceuticals such as lignans and soluble fiber. There is, however, a major drawback of flax as a source of functional food since the seeds contain significant level of cyanogenic glucosides. The final step of cyanogenic glucoside biosynthesis is mediated by UDP-glucose dependent glucosyltransferase. To date, no flax cyanogenic glucosyl transferase genes have been reported with verified biochemical functionality. Here we present a study on the identification and enzymatic characterization of a first flax cyanogenic glucosyltransferase, LuCGT1. We show that LuCGT1 was highly active towards both aliphatic and aromatic substrates. The LuCGT1 gene is expressed in leaf tissues as well as in developing seeds, and its expression level was drastically reduced in flax mutant lines low in cyanogenic glucosides. Identification of LuCGT1 provides a molecular handle for developing gene specific markers for targeted breeding of low cyanogenic glucosides in flax.

SUBMITTER: Kazachkov M

PROVIDER: S-EPMC7001965 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Molecular identification and functional characterization of a cyanogenic glucosyltransferase from flax (Linum unsitatissimum).

Kazachkov Michael M Li Qiang Q Shen Wenyun W Wang Liping L Gao Peng P Xiang Daoquan D Datla Raju R Zou Jitao J

PloS one 20200205 2

Flax seed has become consumers' choice for not only polyunsaturated alpha-linolenic fatty acid but also nutraceuticals such as lignans and soluble fiber. There is, however, a major drawback of flax as a source of functional food since the seeds contain significant level of cyanogenic glucosides. The final step of cyanogenic glucoside biosynthesis is mediated by UDP-glucose dependent glucosyltransferase. To date, no flax cyanogenic glucosyl transferase genes have been reported with verified bioch ...[more]

PMID: 32023283

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Project description:BackgroundFlax (Linum usitatissimum L.) is an important crop for the production of bioproducts derived from its seed and stem fiber. Transposable elements (TEs) are widespread in plant genomes and are a key component of their evolution. The availability of a genome assembly of flax (Linum usitatissimum) affords new opportunities to explore the diversity of TEs and their relationship to genes and gene expression.ResultsFour de novo repeat identification algorithms (PILER, RepeatScout, LTR_finder and LTR_STRUC) were applied to the flax genome assembly. The resulting library of flax repeats was combined with the RepBase Viridiplantae division and used with RepeatMasker to identify TEs coverage in the genome. LTR retrotransposons were the most abundant TEs (17.2% genome coverage), followed by Long Interspersed Nuclear Element (LINE) retrotransposons (2.10%) and Mutator DNA transposons (1.99%). Comparison of putative flax TEs to flax transcript databases indicated that TEs are not highly expressed in flax. However, the presence of recent insertions, defined by 100% intra-element LTR similarity, provided evidence for recent TE activity. Spatial analysis showed TE-rich regions, gene-rich regions as well as regions with similar genes and TE density. Monte Carlo simulations for the 71 largest scaffolds (? 1 Mb each) did not show any regional differences in the frequency of TE overlap with gene coding sequences. However, differences between TE superfamilies were found in their proximity to genes. Genes within TE-rich regions also appeared to have lower transcript expression, based on EST abundance. When LTR elements were compared, Copia showed more diversity, recent insertions and conserved domains than the Gypsy, demonstrating their importance in genome evolution.ConclusionsThe calculated 23.06% TE coverage of the flax WGS assembly is at the low end of the range of TE coverages reported in other eudicots, although this estimate does not include TEs likely found in unassembled repetitive regions of the genome. Since enrichment for TEs in genomic regions was associated with reduced expression of neighbouring genes, and many members of the Copia LTR superfamily are inserted close to coding regions, we suggest Copia elements have a greater influence on recent flax genome evolution while Gypsy elements have become residual and highly mutated.

Project description:BackgroundFlaxseed oil is characterized by high content of essential polyunsaturated fatty acids (PUFA) promoted as a human dietary supplement protecting against atherosclerosis. The disadvantage of the high PUFA content in flax oil is high susceptibility to oxidation, which can result in carcinogenic compound formation. Linola flax cultivar is characterized by high linoleic acid content in comparison to traditional flax cultivars rich in linolenic acid. The changes in fatty acid proportions increase oxidative stability of Linola oil and broaden its use as an edible oil for cooking. However one of investigated transgenic lines has high ALA content making it suitable as omega-3 source. Protection of PUFA oxidation is a critical factor in oil quality. The aim of this study was to investigate the impact of phenylpropanoid contents on the oil properties important during the whole technological process from seed storage to grinding and oil pressing, which may influence health benefits as well as shelf-life, and to establish guidelines for the selection of new cultivars.MethodsThe composition of oils was determined by chromatographic (GS-FID and LC-PDA-MS) methods. Antioxidant properties of secondary metabolites were analyzed by DPPH method. The stability of oils was investigated: a) during regular storage by measuring acid value peroxide value p-anisidine value malondialdehyde, conjugated dienes and trienes; b) by using accelerated rancidity tests by TBARS reaction; c) by thermoanalytical - differential scanning calorimetry (DSC).ResultsIn one approach, in order to increase oil stability, exogenous substances added are mainly lipid soluble antioxidants from the isoprenoid pathway, such as tocopherol and carotene. The other approach is based on transgenic plant generation that accumulates water soluble compounds. Increased accumulation of phenolic compounds in flax seeds was achieved by three different strategies that modify genes coding for enzymes from the phenylpropanoid pathway. The three types of transgenic flax had different phenylpropanoid profiles detected in oil, highly increasing its stability.ConclusionsWe found that hydrophilic phenylpropanoids more than lipophilic isoprenoid compounds determine oil stability however they can work synergistically. Among phenolics the caffeic acid was most effective in increasing oil stability.

Project description:Coexistence allows growers and consumers the choice of producing or purchasing conventional or organic crops with known standards for adventitious presence of genetically engineered (GE) seed. Flax (Linum usitatissimum L.) is multipurpose oilseed crop in which product diversity and utility could be enhanced for industrial, nutraceutical and pharmaceutical markets through genetic engineering. If GE flax were released commercially, pollen-mediated gene flow will determine in part whether GE flax could coexist without compromising other markets. As a part of pre-commercialization risk assessment, we quantified pollen-mediated gene flow between two cultivars of flax. Field experiments were conducted at four locations during 2006 and 2007 in western Canada using a concentric donor (20 × 20 m) receptor (120 × 120 m) design. Gene flow was detected through the xenia effect of dominant alleles of high ?-linolenic acid (ALA; 18:3(cis?9,12,15)) to the low ALA trait. Seeds were harvested from the pollen recipient plots up to a distance of 50 m in eight directions from the pollen donor. High ALA seeds were identified using a thiobarbituric acid test and served as a marker for gene flow. Binomial distribution and power analysis were used to predict the minimum number of seeds statistically required to detect the frequency of gene flow at specific ? (confidence interval) and power (1-?) values. As a result of the low frequency of gene flow, approximately 4 million seeds were screened to derive accurate quantification. Frequency of gene flow was highest near the source: averaging 0.0185 at 0.1 m but declined rapidly with distance, 0.0013 and 0.00003 at 3 and 35 m, respectively. Gene flow was reduced to 50% (O??) and 90% (O??) between 0.85 to 2.64 m, and 5.68 to 17.56 m, respectively. No gene flow was detected at any site or year > 35 m distance from the pollen source, suggesting that frequency of gene flow was ? 0.00003 (P = 0.95). Although it is not possible to eliminate all adventitious presence caused by pollen-mediated gene flow, through harvest blending and the use of buffer zones between GE and conventional flax fields, it could be minimized. Managing other sources of adventitious presence including seed mixing and volunteer populations may be more problematic.

Dataset Information

Molecular identification and functional characterization of a cyanogenic glucosyltransferase from flax (Linum unsitatissimum).

Publications

Molecular identification and functional characterization of a cyanogenic glucosyltransferase from flax (Linum unsitatissimum).

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