Empirical threshold values for quantitative trait mapping.

Tocopherols and tocotrienols, collectively known as tocochromanols, are the major lipid-soluble antioxidants in maize (Zea mays L.) grain. Given that individual tocochromanols differ in their degree of vitamin E activity, variation for tocochromanol composition and content in grain from among diverse maize inbred lines has important nutritional and health implications for enhancing the vitamin E and antioxidant contents of maize-derived foods through plant breeding. Toward this end, we conducted a genome-wide association study of six tocochromanol compounds and 14 of their sums, ratios, and proportions with a 281 maize inbred association panel that was genotyped for 591,822 SNP markers. In addition to providing further insight into the association between ZmVTE4 (γ-tocopherol methyltransferase) haplotypes and α-tocopherol content, we also detected a novel association between ZmVTE1 (tocopherol cyclase) and tocotrienol composition. In a pathway-level analysis, we assessed the genetic contribution of 60 a priori candidate genes encoding the core tocochromanol pathway (VTE genes) and reactions for pathways supplying the isoprenoid tail and aromatic head group of tocochromanols. This analysis identified two additional genes, ZmHGGT1 (homogentisate geranylgeranyltransferase) and one prephenate dehydratase parolog (of four in the genome) that also modestly contribute to tocotrienol variation in the panel. Collectively, our results provide the most favorable ZmVTE4 haplotype and suggest three new gene targets for increasing vitamin E and antioxidant levels through marker-assisted selection.

/pdf/genome-wide-association-study-and-pathway-level-analysis-of-4vohaazl6z.pdf

Genome-Wide Association Study and Pathway-Level Analysis of Tocochromanol Levels in Maize Grain

Tocopherols are lipophilic antioxidants and together with tocotrienols belong to the vitamin-E family. The four forms of tocopherols (α-, β-, γ- and δ-tocopherols) consist of a polar chromanol ring and lipophilic prenyl chain with differences in the position and number of methyl groups. The biosynthesis of tocopherols takes place mainly in plastids of higher plants from precursors derived from two metabolic pathways: homogentisic acid, an intermediate of degradation of aromatic amino acids, and phytyldiphosphate, which arises from methylerythritol phosphate pathway. The regulation of tocopherol biosynthesis in photosynthetic organisms occurs, at least partially, at the level of key enzymes as such including p-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27), homogentisate phytyltransferase (HPT, EC 2.5.1.-), tocopherol cyclase (TC, EC 5.4.99.-), and two methyltransferases. Tocopherol biosynthesis changes during plant development and in response toward different stresses induced by high-intensity light, drought, high salinity, heavy metals, and chilling. It is supposed that scavenging of lipid peroxy radicals and quenching of singlet oxygen are the main functions of tocopherols in photosynthetic organisms. The antioxidant action of tocopherols is related to the formation of tocopherol quinone and its following recycling or degradation. However, until now, the mechanisms of tocopherol degradation in plants have not been established in detail. This review focuses on mechanisms of tocopherols biosynthesis and its regulation in photosynthetic organisms. In addition, available information on tocopherol degradation is summarized.

Tocopherol biosynthesis: chemistry, regulation and effects of environmental factors

Tocopherols, together with tocotrienols and plastochromanols belong to a group of lipophilic compounds also called tocochromanols or vitamin E. Considered to be one of the most powerful antioxidants, tocochromanols are solely synthesized by photosynthetic organisms including plants, algae, and cyanobacteria and, therefore, are an essential component in the human diet. Tocochromanols potent antioxidative properties are due to their ability to interact with polyunsaturated acyl groups and scavenge lipid peroxyl radicals and quench reactive oxygen species (ROS), thus protecting fatty acids from lipid peroxidation. In the plant model species Arabidopsis thaliana, the required genes for tocopherol biosynthesis and functional roles of tocopherols were elucidated in mutant and transgenic plants. Recent research efforts have led to new outcomes for the vitamin E biosynthetic and related pathways, and new possible alternatives for the biofortification of important crops have been suggested. Here, we review 30 years of research on tocopherols in model and crop species, with emphasis on the improvement of vitamin E content using transgenic approaches and classical breeding. We will discuss future prospects to further improve the nutritional value of our food.

/pdf/recent-advances-in-our-understanding-of-tocopherol-1i3bd0epm5.pdf

Recent Advances in our Understanding of Tocopherol Biosynthesis in Plants: An Overview of Key Genes, Functions, and Breeding of Vitamin E Improved Crops.

Madagascar periwinkle (Catharanthus roseus) is the sole source of the anticancer drugs vinblastine and vincristine, bisindole alkaloids derived from the dimerization of the terpenoid indole alkaloids vindoline and catharanthine. Full elucidation of the biosynthetic pathways of these compounds is a prerequisite for metabolic engineering efforts that will improve production of these costly molecules. However, despite the medical and commercial importance of these natural products, the biosynthetic pathways remain poorly understood. Here we report the identification and characterization of a C. roseus cDNA encoding an S-adenosyl-L-methionine-dependent N methyltransferase that catalyzes a nitrogen methylation involved in vindoline biosynthesis. Recombinant enzyme produced in Escherichia coli is highly substrate specific, displaying a strict requirement for a 2,3-dihydro bond in the aspidosperma skeleton. The corresponding gene transcript is induced in methyl jasmonate-elicited seedlings, along with the other known vindoline biosynthetic transcripts. Intriguingly, this unique N methyltransferase is most similar at the amino acid level to the plastidic γ-tocopherol C methyltransferases of vitamin E biosynthesis, suggesting an evolutionary link between these two functionally disparate methyltransferases.

https://www.pnas.org/content/pnas/107/44/18793.full.pdf

Homolog of tocopherol C methyltransferases catalyzes N methylation in anticancer alkaloid biosynthesis

Rapeseed (Brassica napus L.) is the most important oil crop of temperate climates. Rapeseed oil contains tocopherols, also known as vitamin E, which is an indispensable nutrient for humans and animals due to its antioxidant and radical scavenging abilities. Moreover, tocopherols are also important for the oxidative stability of vegetable oils. Therefore, seed oil with increased tocopherol content or altered tocopherol composition is a target for breeding. We investigated the role of nucleotide variations within candidate genes from the tocopherol biosynthesis pathway. Field trials were carried out with 229 accessions from a worldwide B. napus collection which was divided into two panels of 96 and 133 accessions. Seed tocopherol content and composition were measured by HPLC. High heritabilities were found for both traits, ranging from 0.62 to 0.94. We identified polymorphisms by sequencing selected regions of the tocopherol genes from the 96 accession panel. Subsequently, we determined the population structure (Q) and relative kinship (K) as detected by genotyping with genome-wide distributed SSR markers. Association studies were performed using two models, the structure-based GLM+Q and the PK mixed model. Between 26 and 12 polymorphisms within two genes (BnaX.VTE3.a, BnaA.PDS1.c) were significantly associated with tocopherol traits. The SNPs explained up to 16.93 % of the genetic variance for tocopherol composition and up to 10.48 % for total tocopherol content. Based on the sequence information we designed CAPS markers for genotyping the 133 accessions from the 2nd panel. Significant associations with various tocopherol traits confirmed the results from the first experiment. We demonstrate that the polymorphisms within the tocopherol genes clearly impact tocopherol content and composition in B. napus seeds. We suggest that these nucleotide variations may be used as selectable markers for breeding rapeseed with enhanced tocopherol quality.

/pdf/a-candidate-gene-based-association-study-of-tocopherol-4a9642jfhh.pdf

A candidate gene-based association study of tocopherol content and composition in rapeseed (Brassica napus).

Background Tocopherols are important antioxidants in vegetable oils; when present as vitamin E, tocopherols are an essential nutrient for humans and livestock. Rapeseed (Brassica napus L, AACC, 2 n = 38) is one of the most important oil crops and a major source of tocopherols. Although the tocopherol biosynthetic pathway has been well elucidated in the model photosynthetic organisms Arabidopsis thaliana and Synechocystis sp. PCC6803, knowledge about the genetic basis of tocopherol biosynthesis in seeds of rapeseed is scant. This project was carried out to dissect the genetic basis of seed tocopherol content and composition in rapeseed through quantitative trait loci (QTL) detection, genome-wide association analysis, and homologous gene mapping. Methodology/principal findings We used a segregating Tapidor × Ningyou7 doubled haploid (TNDH) population, its reconstructed F(2) (RC-F(2)) population, and a panel of 142 rapeseed accessions (association panel). Genetic effects mainly contributed to phenotypic variations in tocopherol content and composition; environmental effects were also identified. Thirty-three unique QTL were detected for tocopherol content and composition in TNDH and RC-F(2) populations. Of these, seven QTL co-localized with candidate sequences associated with tocopherol biosynthesis through in silico and linkage mapping. Several near-isogenic lines carrying introgressions from the parent with higher tocopherol content showed highly increased tocopherol content compared with the recurrent parent. Genome-wide association analysis was performed with 142 B. napus accessions. Sixty-one loci were significantly associated with tocopherol content and composition, 11 of which were localized within the confidence intervals of tocopherol QTL. Conclusions/significance This joint QTL, candidate gene, and association mapping study sheds light on the genetic basis of seed tocopherol biosynthesis in rapeseed. The sequences presented here may be used for marker-assisted selection of oilseed rape lines with superior tocopherol content and composition.

/pdf/unraveling-the-genetic-basis-of-seed-tocopherol-content-and-33gsrs1xkb.pdf

Unraveling the genetic basis of seed tocopherol content and composition in rapeseed (Brassica napus L.).

Rapeseed (Brassica napus L.) is one of the major oilseed crops and an important source for tocopherols known as vitamin E in human nutrition. Increasing the tocopherol content and altering the tocopherol composition is a major goal in rapeseed breeding. The genes encoding enzymes from the tocopherol pathway have been cloned from model species. However, only scant data about tocopherol genes from crop species have been available. We have cloned four sequences of a gene family from B. napus with homology to the Arabidopsis thalianaVTE4 gene. The sequences were amplified by PCR with primers derived from the A. thaliana gene. BAC-clones were isolated to analyze the genomic structure of the BnaX.VTE4-loci. In contrast to the A. thaliana gene all B. napus sequences have two additional introns. For functional analysis, the BnaA.VTE4.a1 sequence was transformed into A. thaliana. Seeds from transgenic offspring showed a 50-fold increase of the α-tocopherol fraction which is in accordance with the predicted function of the gene. A marker assay was established and the BnaA.VTE4.a1 sequence was mapped to the end of chromosome A02 of the Tapidor × Ningyou7 genetic map, where also two QTL for α-tocopherol content had been mapped. Thus, the BnaA.VTE4.a1 gene is a promising candidate for these QTL and can be used for marker assisted selection for α-tocopherol content in rapeseed.

Genetic mapping, cloning, and functional characterization of the BnaX.VTE4 gene encoding a γ-tocopherol methyltransferase from oilseed rape

Brassica napus L. is one of the most important oilseed crops in the world. Improving oil quality by increasing vitamin E content is a major target of rapeseed breeding. Vitamin E compounds, or tocopherols, are lipid-soluble antioxidants that are essential nutrients for mammals. In this study, we report the characterization of rapeseed orthologs of the Arabidopsis tocopherol genes VTE1, VTE2 and PDS1. For each gene, at least two homologous sequences were found, and their expression was analysed in different tissues from three rapeseed genotypes. Genetic complementation experiments demonstrated that BnaX.VTE1.b and BnaA. VTE2.b homologs are capable of recovering seed gamma-tocopherol in Arabidopsis mutants. Overexpression of the genes in Arabidopsis Col-0 shifted the seed tocopherol composition towards higher alpha-tocopherol without increasing the total tocopherol content. To address the functionality of BnPDS1 sequences, we performed overexpression tests in Escherichia coli and enzymatic activity analyses. Overall, our results show that the identified sequences from rapeseed are functional orthologs of the Arabidopsis VTE genes and thus have considerable potential as molecular markers for selecting rapeseed with improved seed oil quality.

Jessica Endrigkeit

Papers

A candidate gene-based association study of tocopherol content and composition in rapeseed (Brassica napus).

Unraveling the genetic basis of seed tocopherol content and composition in rapeseed (Brassica napus L.).

Genetic mapping, cloning, and functional characterization of the BnaX.VTE4 gene encoding a γ-tocopherol methyltransferase from oilseed rape

Genetic and functional analysis of tocopherol biosynthesis pathway genes from rapeseed (Brassica napus L.)