Homogentisate Phytyltransferase Research Articles

Heterologous Expression of Sunflower HaHPT and HaTMT Genes Enhances Rice-Grain Vitamin E Content.

Insufficient dietary vitamin intake can lead to severe health conditions in humans. Improving the vitamin E (VE) content of food crops such as rice through breeding is an economical and effective means to alleviate this problem. In this study, Homogentisate phytyltransferase (HPT) and γ-tocopherol methyltransferase (γ-TMT), two genes derived from sunflower (Helianthus annuus L., a high VE species), were introduced into an elite rice (Oryza sativa L.) cultivar "Ningjing 7" for biofortification. We verified the successful expression of the two genes in multiple transformation events. High-performance liquid chromatography revealed that transgenic plants expressing either HaHPT alone or HaHPT and HaTMT accumulate more VE compared with the wild type. We also revealed that the level of α-tocopherol, the form of VE with the highest biological activity, had increased to 2.33 times in transgenic HaTMT plants compared with the wild type. Transcriptome analysis revealed that the expression levels of some chlorophyll synthesis pathway genes related to VE precursor synthesis significantly increased during grain filling in transgenic rice grains. No difference in agronomic traits was observed between the transgenic plants and their wild type except for a slightly reduced plant height associated with the transgenic plants. These data demonstrate that the heterologous expression of HaHPT gene is effective in increasing the total VE content, while HaTMT plays an important role in the relative abundance of α-tocopherol in rice grains. This study demonstrates a promising strategy for breeding rice with elevated VE content via metabolic engineering.

Vitamin E biofortification: enhancement of seed tocopherol concentrations by altered chlorophyll metabolism.

Homogentisate Phytyltransferase (HPT) catalyzes condensation of homogentisate (HGA) and phytyl diphosphate (PDP) to produce tocopherols, but can also synthesize tocotrienols using geranylgeranyl diphosphate (GGDP) in plants engineered for deregulated HGA synthesis. In contrast to prior tocotrienol biofortification efforts, engineering enhanced tocopherol concentrations in green oilseeds has proven more challenging due to the integral role of chlorophyll metabolism in supplying the PDP substrate. This study show that RNAi suppression of CHLSYN coupled with HPT overexpression increases tocopherol concentrations by >two-fold in Arabidopsis seeds. We obtained additional increases in seed tocopherol concentrations by engineering increased HGA production via overexpression of bacterial TyrA that encodes chorismate mutase/prephenate dehydrogenase activities. In overexpression lines, seed tocopherol concentrations increased nearly three-fold, and resulted in modest tocotrienol accumulation. We further increased total tocochromanol concentrations by enhancing production of HGA and GGDP by overexpression of the gene for hydroxyphenylpyruvate dioxygenase (HPPD). This shifted metabolism towards increased amounts of tocotrienols relative to tocopherols, which was reflected in corresponding increases in ratios of GGDP/PDP in these seeds. Overall, our results provide a theoretical basis for genetic improvement of total tocopherol concentrations in green oilseeds (e.g., rapeseed, soybean) through strategies that include seed-suppression of CHLSYN coupled with increased HGA production.

Transcriptional and protein structural characterization of homogentisate phytyltransferase genes in barley, wheat, and oat

BackgroundHomogentisate phytyltransferase (HPT) is the critical enzyme for the biosynthesis of tocopherols (vitamin E), which are the major lipid-soluble antioxidants and help plants adapt to various stress conditions. HPT is generally strictly conserved in various plant genomes; however, a divergent lineage HPT2 was identified recently in some Triticeae species. The molecular function and transcriptional profiles of HPT2 remain to be characterized.ResultsIn this study, we performed comprehensive transcriptome data mining of HPT1 and HPT2 in different tissues and stages of barley (Hordeum vulgare), wheat (Triticum aestivum), and oat (Avena sativa), followed by qRT-PCR experiments on HPT1 and HPT2 in different tissues of barley and wheat. We found that the common HPT1 genes (HvHPT1, TaHPT1s, and AsHPT1s) displayed a conserved transcriptional pattern in the three target species and were universally transcribed in various tissues, with a notable preference in leaf. In contrast, HPT2 genes (HvHPT2, TaHPT2, and AsHPT2) were specifically transcribed in spike (developmentally up-regulated) and shoot apex tissues, displaying a divergent tissue-specific pattern. Cis-regulatory elements prediction in the promoter region identified common factors related to light-, plant hormone-, low temperature-, drought- and defense- responses in both HPT1s and HPT2s. We observed the transcriptional up-regulation of HvHPT1 and HvHPT2 under various stress conditions, supporting their conserved function in environmental adaption. We detected a clear, relaxed selection pressure in the HPT2 lineage, consistent with the predicted evolution pattern following gene duplication. Protein structural modelling and substrate docking analyses identified putative catalytic amino acid residues for HvHPT1 and HvHPT2, which are strictly conserved and consistent with their function in vitamin E biosynthesis.ConclusionsWe confirmed the presence of two lineages of HPT in Triticeae and Aveninae, including hexaploid oat, and characterized their transcriptional profiles based on transcriptome and qRT-PCR data. HPT1s were ubiquitously transcribed in various tissues, whilst HPT2s were highly expressed in specific stages and tissue. The active transcription of HPT2s, together with its conserved cis-elements and protein structural features, support HPT2s’ role in tocopherol production in Triticeae. This study is the first protein structural analysis on the membrane-bound plant HPTs and provides valuable insights into its catalytic mechanism.

Genetic improvement of tocotrienol content enhances the oxidative stability of canola oil.

Tocotrienols and tocopherols, which are synthesized in plastids of plant cells with similar functionalities, comprise vitamin E to serve as a potent lipid-soluble antioxidant in plants. The synthesis of tocopherols involves the condensation of homogentisic acid (HGA) and phytyl diphosphate (PDP) under the catalysis of homogentisate phytyltransferase (HPT). Tocotrienol synthesis is initiated by the condensation of HGA and geranylgeranyl diphosphate (GGDP) mediated by homogentisate geranylgeranyl transferase (HGGT). As one of the most important oil crops, canola seed is regarded as an ideal plant to efficiently improve the production of vitamin E tocochromanols through genetic engineering approaches. However, only a modest increase in tocopherol content has been achieved in canola seed to date. In this study, we transformed barley HGGT (HvHGGT) into canola to improve total tocochromanol content in canola seeds. The results showed that the total tocochromanol content in the transgenic canola seeds could be maximally increased by fourfold relative to that in wild-type canola seeds. Notably, no negative impact on important agronomic traits was observed in transgenic canola plants, indicating great application potential of the HvHGGT gene in enhancing tocochromanol content in canola in the future. Moreover, the oil extracted from the transgenic canola seeds exhibited significantly enhanced oxidative stability under high temperature in addition to the increase in total tocochromanol content, demonstrating multiple desirable properties of HvHGGT.

Comparative gene retention analysis in barley, wild emmer, and bread wheat pangenome lines reveals factors affecting gene retention following gene duplication

BackgroundGene duplication is a prevalent phenomenon and a major driving force underlying genome evolution. The process leading to the fixation of gene duplicates following duplication is critical to understand how genome evolves but remains fragmentally understood. Most previous studies on gene retention are based on gene duplicate analyses in single reference genome. No population-based comparative gene retention analysis has been performed to date.ResultsTaking advantage of recently published genomic data in Triticeae, we dissected a divergent homogentisate phytyltransferase (HPT2) lineage caught in the middle stage of gene fixation following duplication. The presence/absence of HPT2 in barley (diploid), wild emmer (tetraploid), and bread wheat (hexaploid) pangenome lines appears to be associated with gene dosage constraint and environmental adaption. Based on these observations, we adopted a phylogeny-based orthology inference approach and performed comparative gene retention analyses across barley, wild emmer, and bread wheat. This led to the identification of 326 HPT2-pattern-like genes at whole genome scale, representing a pool of gene duplicates in the middle stage of gene fixation. Majority of these HPT2-pattern-like genes were identified as small-scale duplicates, such as dispersed, tandem, and proximal duplications. Natural selection analyses showed that HPT2-pattern-like genes have experienced relaxed selection pressure, which is generally accompanied with partial positive selection and transcriptional divergence. Functional enrichment analyses showed that HPT2-pattern-like genes are over-represented with molecular-binding and defense response functions, supporting the potential role of environmental adaption during gene retention. We also observed that gene duplicates from larger gene family are more likely to be lost, implying a gene dosage constraint effect. Further comparative gene retention analysis in barley and bread wheat pangenome lines revealed combined effects of species-specific selection and gene dosage constraint.ConclusionsComparative gene retention analyses at the population level support gene dosage constraint, environmental adaption, and species-specific selection as three factors that may affect gene retention following gene duplication. Our findings shed light on the evolutionary process leading to the retention of newly formed gene duplicates and will greatly improve our understanding on genome evolution via duplication.

Enhanced vitamin E content in an Indica rice cultivar harbouring two transgenes from Arabidopsis thaliana involved in tocopherol biosynthesis pathway.

Micronutrient deficiency results in malnutrition, which is prevalent all over the world and may lead to premature death in women and children (White and Broadley, 2009). Strategies formulated earlier including supplementation and fortified foods were not successful, owing to socio-economic and technical hurdles (Mayer et al. 2008). The subsequently evolved strategy of bio-fortification is a viable biotechnological tool to achieve desired results without compromising the agronomical values of crops. Conferring the genetic trait to improve vital nutrient accumulation in the edible parts of staple food crops, such as rice, through metabolic engineering is considered a fast, sustainable, and cost-effective alternative to conventional breeding (Maestre et al. 2017). In earlier reports from our lab, enhanced α-tocopherol levels in the stable transformants of Nicotiana tabacum (Harish et al. 2013a, b) and in Nicotiana benthamiana adopting a transient expression system using A. thaliana tocopherol cyclase (TC) and homogentisate phytyl transferase (HPT) (Sathish et al. 2018) were shown. In the present study, Agrobacterium-mediated transformation of Indica rice ASD16 with two genes involved in tocopherol biosynthesis, viz., TC and HPT were carried out and the transgenic plants were analyzed for the vitamin E (α- tocopherol) content.

Interplay between antioxidants in response to photooxidative stress in Arabidopsis

Tocochromanols (tocopherols, tocotrienols and plastochromanol-8), isoprenoid quinone (plastoquinone-9 and plastoquinol-9) and carotenoids (carotenes and xanthophylls), are lipid-soluble antioxidants in the chloroplasts, which play an important defensive role against photooxidative stress in plants. In this study, the interplay between the antioxidant activities of those compounds in excess light stress was analyzed in wild-type (WT) Arabidopsis thaliana and in a tocopherol cyclase mutant (vte1), a homogentisate phytyl transferase mutant (vte2) and a tocopherol cyclase overexpressor (VTE1oex). The results reveal a strategy of cooperation and replacement between α-tocopherol, plastochromanol-8, plastoquinone-9/plastoquinol-9 and zeaxanthin. In the first line of defense (non-radical mechanism), singlet oxygen is either physically or chemically quenched by α-tocopherol; however, when α-tocopherol is consumed, zeaxanthin and plastoquinone-9/plastoquinol-9 can provide alternative protection against singlet oxygen toxicity by functional replacement of α-tocopherol either by zeaxanthin for the physical quenching or by plastoquinone-9/plastoquinol-9 for the chemical quenching. When singlet oxygen escapes this first line of defense, it oxidizes lipids and forms lipid hydroperoxides, which are oxidized to lipid peroxyl radicals by ferric iron. In the second line of defense (radical mechanism), lipid peroxyl radicals are scavenged by α-tocopherol. After its consumption, plastochromanol-8 overtakes this function. We provide a comprehensive description of the reaction pathways underlying the non-radical and radical antioxidant activities of α-tocopherol, carotenoids, plastoquinone-9/plastoquinol-9 and plastochromanol-8. The interplay between the different plastid lipid-soluble antioxidants in the non-radical and the radical mechanism provides step by step insights into protection against photooxidative stress in higher plants.

The progress of the biosynthesis of vitamin E

Vitamin E is a fat-soluble compound with powerful antioxidative and anticancer activity, which constitutes eight isoforms, namely α-, β-, γ-, δ-tocopherol and α-, β-, γ-, δ-tocotrienol. With the wide use of vitamin E in food, medicine, cosmetics and other fields, the demand of vitamin E in market is huge. Currently in the market, vitamin E is about 80% from chemical synthesis and 20% from extraction of oil distillates derived from plants or seeds. However, chemical synthesis of vitamin E suffered from the use of toxic catalysts, making the process environmentally unfriendly and unsustainable. In addition, the chemical synthesis technologies mainly synthesized the racemate of vitamin E and the enantiomerically pure vitamin E is mostly extracted from limited agricultural resources. Recently, using microorganisms to heterologously express metabolic pathways originated from plants is an attractive strategy due to the environmentally friendly, economic and easily scalable. Moreover, the progress of synthetic biology and metabolic engineering enabled the genetically tractable microorganisms such as Escherichia coli and Saccharomyces cerevisiae to successfully produce many natural compounds. As the natural pathways and related enzymes to synthesize vitamin E have been found in the last decades, it is feasible to engineer microorganisms to produce vitamin E. δ-tocotrienol, one isoform of vitamin E, was de novo heterologously synthesized in Escherichia coli and Saccharomyces cerevisiae with the production of 15 μg/(g cdw) and the titer of 4.10 mg/L (1.3 mg/(g cdw)), respectively. In this review, we firstly summarized the natural pathways and the recent progress on the enhanced biosynthesis of Vitamin E. Then the pathways and the recent advances on metabolically engineering strategies for the enhanced key intimidates (homogentisate and geranylgeranyl diphosphate) in industrial model microorganism was summarized. Specifically, we reviewed the advances on the shikimate pathway and the 2-C-methyl-d-erythritol-4-phosphate (MEP) and mevalonic acid (MVA) pathway in different chassis. Finally, we summarized the present situation and prospected the potential of biosynthesis of vitamin E in microorganisms. Based on the current researches on the improving production of vitamin E in microorganisms, in addition to enhance the supply of the precursors (homogentisate and geranylgeranyl diphosphate) by metabolic engineering, more attention needs to be paid to improve the enzyme activities of the key enzymes like homogentisate phytyl transferase (HPT) and tocopherol/tocotrienol cyclase (TC), which restricted the conversion rate of the precursors to the target product vitamin E. In the future, biotechnology such as genetic engineering, metabolic engineering and synthetic biology can be combined with the natural pathway of vitamin E to biosynthesize specific monomer or mixture of vitamin E in different model microorganisms. To further improve the production of vitamin E in the engineered microorganism, diverse genes and enzymes from multiple species involved in the biosynthesis of vitamin E needs to be characterized and functionalized. Enzyme engineering could also be used to improve the enzyme activities and consequently enhance the production of vitamin E.

Genome wide association mapping for tocopherol concentration in soybean seeds across multiple environments

Tocopherol (Toc) in soybean seeds has value for foods, medicines, cosmetics, and animal husbandry. Soybean breeders and soybean-derived food industries have thus focused on breeding new soybean cultivars with higher seed Toc content. In this study, an association panel consisting of 180 diverse soybean accessions were used to detect quantitative trait nucleotides (QTNs) and scan possible candidate genes based on genome-wide association analysis (GWAS) and high-throughput single-nucleotide polymorphisms (SNP). Approximately 22,611 SNPs were identified using the specific locus amplified fragment sequencing (SLAF-seq) approach and based on minor allele frequencies (MAFs) > 5 %, which spanned 84.10 % of the soybean whole genome. For the three individuals including α-tocopherol (α-Toc), γ- tocopherol (γ-Toc), δ- tocopherol (δ-Toc), and total tocopherol (total Toc) levels, a total of 18 quantitative trait nucleotides (QTNs) distributed on the 18 genomic regions of 10 chromosomes were verified via GWAS. Seven QTNs (rs55811336, rs7975863, rs37094897, rs7097743, rs17125409, rs330000, and rs4730199) could be identified in more than three environments, and the other eleven could be identified in least two environments. A total of 6, 8, 4, and 10 QTNs were novel loci for α-Toc, γ-Toc, δ-Toc, and total Toc, respectively. Nine QTNs (rs37505279, rs37686178, rs36359429, rs6898494, rs7097743, rs17125409, rs10200201, rs21080726, and rs5680781) were associated with more than one Toc component. A total of 216 candidate genes near the 200-kb flanking region of these identified QTNs were identified as potential candidate genes associated with soybean seed Toc levels. Of these genes, Glyma.17G061900 (located near QTN rs4730199 on Chr.17), a homogentisate phytyltransferase 1 that catalytic homogentisate to 2-methyl-6-phyl-1,4-benzoquinol in Toc biosynthesis, was detected through association analysis, haplotype analysis, and expression pattern analysis. The identified QTNs with beneficial alleles and the candidate genes would be valuable for improving Toc levels in soybean seeds and improving the nutritional value of soybean oil.

Enhancing flavonoid production by promiscuous activity of prenyltransferase, BrPT2 from Boesenbergia rotunda.

Flavonoids and prenylated flavonoids are active components in medicinal plant extracts which exhibit beneficial effects on human health. Prenylated flavonoids consist of a flavonoid core with a prenyl group attached to it. This prenylation process is catalyzed by prenyltranferases (PTs). At present, only a few flavonoid-related PT genes have been identified. In this study, we aimed to investigate the roles of PT in flavonoid production. We isolated a putative PT gene (designated as BrPT2) from a medicinal ginger, Boesenbergia rotunda. The deduced protein sequence shared highest gene sequence homology (81%) with the predicted homogentisate phytyltransferase 2 chloroplastic isoform X1 from Musa acuminata subsp. Malaccensis. We then cloned the BrPT2 into pRI vector and expressed in B. rotunda cell suspension cultures via Agrobacterium-mediated transformation. The BrPT2-expressing cells were fed with substrate, pinostrobin chalcone, and their products were analyzed by liquid chromatography mass spectrometry. We found that the amount of flavonoids, namely alpinetin, pinostrobin, naringenin and pinocembrin, in BrPT2-expressing cells was higher than those obtained from the wild type cells. However, we were unable to detect any targeted prenylated flavonoids. Further in-vitro assay revealed that the reaction containing the BrPT2 protein produced the highest accumulation of pinostrobin from the substrate pinostrobin chalcone compared to the reaction without BrPT2 protein, suggesting that BrPT2 was able to accelerate the enzymatic reaction. The finding of this study implied that the isolated BrPT2 may not be involved in the prenylation of pinostrobin chalcone but resulted in high yield and production of other flavonoids, which is likely related to enzyme promiscuous activities.

Deciphering the genetic basis for vitamin E accumulation in leaves and grains of different barley accessions

Tocopherols and tocotrienols, commonly referred to as vitamin E, are essential compounds in food and feed. Due to their lipophilic nature they protect biomembranes by preventing the propagation of lipid-peroxidation especially during oxidative stress. Since their synthesis is restricted to photosynthetic organisms, plant-derived products are the major source of natural vitamin E. In the present study the genetic basis for high vitamin E accumulation in leaves and grains of different barley (Hordeum vulgare L.) accessions was uncovered. A genome wide association study (GWAS) allowed the identification of two genes located on chromosome 7H, homogentisate phytyltransferase (HPT-7H) and homogentisate geranylgeranyltransferase (HGGT) that code for key enzymes controlling the accumulation of tocopherols in leaves and tocotrienols in grains, respectively. Transcript profiling showed a correlation between HPT-7H expression and vitamin E content in leaves. Allele sequencing allowed to decipher the allelic variation of HPT-7H and HGGT genes corresponding to high and low vitamin E contents in the respective tissues. Using the obtained sequence information molecular markers have been developed which can be used to assist smart breeding of high vitamin E barley varieties. This will facilitate the selection of genotypes more tolerant to oxidative stress and producing high-quality grains.

Full-Length Transcriptome Analysis of the Genes Involved in Tocopherol Biosynthesis in Torreya grandis.

The seeds of Torreya grandis (Cephalotaxaceae) are rich in tocopherols, which are essential components of the human diet as a result of their function in scavenging reactive oxygen and free radicals. Different T. grandis cultivars (10 cultivars selected in this study were researched, and their information is shown in Table S1 of the Supporting Information) vary enormously in their tocopherol contents (0.28-11.98 mg/100 g). However, little is known about the molecular basis and regulatory mechanisms of tocopherol biosynthesis in T. grandis kernels. Here, we applied single-molecule real-time (SMRT) sequencing to T. grandis (X08 cultivar) for the first time and obtained a total of 97 211 full-length transcripts. We proposed the biosynthetic pathway of tocopherol and identified eight full-length transcripts encoding enzymes potentially involved in tocopherol biosynthesis in T. grandis. The results of the correlation analysis between the tocopherol content and gene expression level in the 10 selected cultivars and different kernel developmental stages of the X08 cultivar suggested that homogentisate phytyltransferase coding gene ( TgVTE2b) and γ-tocopherol methyltransferase coding gene ( TgVTE4) may be key players in tocopherol accumulation in the kernels of T. grandis. Subcellular localization assays showed that both TgVTE2b and TgVTE4 were localized to the chloroplast. We also identified candidate regulatory genes similar to WRI1 and DGAT1 in Arabidopsis that may be involved in the regulation of tocopherol biosynthesis. Our findings provide valuable genetic information for T. grandis using full-length transcriptomic analysis, elucidating the candidate genes and key regulatory genes involved in tocopherol biosynthesis. This information will be critical for further molecular-assisted screening and breeding of T. grandis genotypes with high tocopherol contents.

Exogenous 24-epibrassinolide enhanced benzene detoxification in Chlorophytum comosum via overexpression and conjugation by glutathione

Benzene, a hydrophobic xenobiotic, induces cell damage in both humans and plants. Due to its volatilization, benzene is an airborne environmental problem. The potential of an exogenous bioactive brassinosteroid phytohormone to enhance benzene removal for phytoremediation was investigated. Chlorophytum comosum had higher brassinosteroids content under benzene stress. Plant treated with 24-epibrassinolide (EBR) removed significantly more gaseous benzene than untreated plants under both light and dark conditions at an initial benzene of 12.75 μmol in the systematic chambers (P < 0.05). Although benzene increased malondialdehyde in plant tissue, EBR-treated plants lowered this lipid peroxidation by enhancing their antioxidant content and increasing benzene detoxification-related genes expression, including ascorbic acid (AsA), homogentisate phytyltransferase (HPT), and glutathione synthethase (GS). This contributed to maintaining higher photosynthetic performances. Moreover, EBR-treated plants had higher gene expression of ferredoxin–NADP reductase (FNR) and glucose-6-phosphate 1-dehydrogenase (G6PDH), thus promoting NADPH biosynthesis to cope with benzene under light and dark conditions, respectively. Further, higher glutathione biosynthesis promoted more glutathione conjugate of benzene products including S-phenylcysteine (SPC) in EBR-treated plants. Hence, application of exogenous EBR as foliar spray provided for enhanced benzene detoxification via antioxidant content, benzene detoxification-related genes and benzene conjugation products with glutathione (GSH) and consequently greater gaseous benzene removal.

Antioxidant Balance and Regulation in Tomato Genotypes of Different Color

Antioxidants, antioxidant capacity, and the expression of isoprenoid metabolism–related genes and two pigmentation-related transcription factors were studied in four native and four hybrid tomato (Solanum lycopersicum) genotypes with different-colored fruit. Red fruit genotypes were associated with greater lycopene, β-carotene, lipophilic antioxidant capacity, and greater chromoplast-specific lycopene β-cyclase (CYC-B) transcript levels. Orange fruit genotypes had greater concentrations of tocopherols and greater transcript levels of homogentisate phytyl transferase (VTE-2), 1-deoxy-D-xylulose phosphate synthase (DXS), and 4-hydroxyphenylpyruvate dioxygenase (HPPD). The yellow fruit genotype was greater in total polyphenol and hydrophilic antioxidant capacity with greater expression of geranylgeranyl reductase (GGDR), phytol kinase (VTE-5), phytoene synthase (PSY) 2, lycopene β-cyclase (LCY-B), SlNAC1, and SINAC4. Greater levels of individual antioxidants were associated with specific coloration of tomato fruit. Moreover, the negative correlations between the expression of PSY1 and VTE-5, and between lycopene and chlorophyll, suggest a balance between carotenoids, tocopherols, and chlorophylls. The results of this study support either the direct commercialization of tomatoes with different color fruit or use of their genotypes in breeding programs to increase antioxidant levels among existing cultivars.

Rapid enhancement of α-tocopherol content in Nicotiana benthamiana by transient expression of Arabidopsis thaliana Tocopherol cyclase and Homogentisate phytyl transferase genes.

Agrobacterium-mediated transient gene expression have become a method of choice over stable plant genetic transformation. Tocopherols are a family of vitamin E compounds, which are categorized along with tocotrienols occurring naturally in vegetable oils, nuts and leafy green vegetables. This is the first report involving AtTC and AtHPT transient expression in Nicotiana benthamiana and this system can be used efficiently for large scale production of vitamin E. Agroinfiltration studies were carried out in N.benthamiana for the expression of Arabidopsis thaliana (At) genes encoding homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) individually and in combination (HPT + TC). The transgene presence was analyzed by reverse transcription PCR, which showed the presence of both the vitamin E biosynthetic pathway genes. The gene expression analysis was carried out by (reverse transcription quantitative real-time polymerase chain reaction) RT-qPCR and α-tocopherol content was quantified using high performance liquid chromatography (HPLC). The relative gene expression analysis by RT-qPCR confirmed an increased expression pattern where TC + HPT combination recorded the highest of 231 fold, followed by TC gene with 186 fold, whereas the HPT gene recorded 178 fold. The α-tocopherol content in leaves expressing HPT, TC, and HPT + TC was increased by 4.2, 5.9 and 11.3 fold, respectively, as compared to the control. These results indicate that the transient expression of HPT and TC genes has enhanced the vitamin E levels and stable expression of both A. thaliana genes could be an efficient strategy to enhance vitamin E biosynthesis in agricultural crop breeding.

Transcriptional Regulation of Vitamin E Biosynthesis during Germination of Dwarf Fan Palm Seeds.

Vitamin E, a potent antioxidant either presents in the form of tocopherols and/or tocotrienols depending on the plant species, tissue and developmental stage, plays a major role in protecting lipids from oxidation in seeds. Unlike tocopherols, which have a more universal distribution, the occurrence of tocotrienols is limited primarily to monocot seeds. Dwarf fan palm (Chamaerops humilis var. humilis) seeds accumulate tocotrienols in quiescent and dormant seeds, while tocopherols are de novo synthesized during germination. Here, we aimed to elucidate whether tocopherol biosynthesis is regulated at the transcriptional level during germination in this species. We identified and quantified the expression levels of five genes involved in vitamin E biosynthesis, including TYROSINE AMINOTRANSFERASE (ChTAT), HOMOGENTISATE PHYTYLTRANSFERASE (ChHPT), HOMOGENTISATE GERANYLGERANYL TRANSFERASE (ChHGGT), TOCOPHEROL CYCLASE (ChTC) and TOCOPHEROL γ-METHYLTRANSFERASE (Chγ-TMT). Furthermore, we evaluated to what extent variations in the endogenous contents of hormones and hydrogen peroxide (H2O2) correlated with transcriptional regulation. Results showed an increase of ChTAT and ChHPT levels during seed germination, which correlated with an increase of jasmonic acid (JA), gibberellin4 (GA4), and H2O2 contents, while ChHGGT and Chγ-TMT expression levels decreased, thus clearly indicating vitamin E biosynthesis is diverted to tocopherols rather than to tocotrienols. Exogenous application of jasmonic acid increased tocopherol, but not tocotrienol content, thus confirming its regulatory role in vitamin E biosynthesis during seed germination. It is concluded that the biosynthesis of vitamin E is regulated at the transcriptional level during germination in dwarf fan palm seeds, with ChHPT playing a key role in the diversion of the vitamin E pathway towards tocopherols instead of tocotrienols.

High-density linkage mapping of vitamin E content in maize grain

Vitamin E refers to eight distinct compounds collectively known as tocochromanols and can be further divided into two classes, tocotrienols and tocopherols. Tocochromanols are the major lipid-soluble antioxidants in maize (Zea mays L.) grain. Enhancing vitamin E content of maize through plant breeding has important implications for human and animal nutrition. Four inbred lines exhibiting unique variation for tocochromanol compounds were chosen from the Goodman maize diversity panel to construct two biparental mapping populations (N6xNC296 and E2558xCo125). The N6xNC296 population was developed to analyze segregation for α-tocopherol and α-tocotrienol content. The E2558WxCo125 population was developed to analyze segregation for the ratio of total tocotrienols to tocopherols. The tocochromanol variation in two replicates of each population was quantified using liquid chromatography-diode array detection. Using high-density linkage mapping, novel quantitative trait loci (QTL) in the N6xNC296 population were mapped using tocopherol ratio traits. These QTL contain the candidate gene homogentisate phytyltransferase (ZmVTE2) within the respective support intervals. This locus was not mapped in a previous genome-wide association study that analyzed tocochromanols in the Goodman diversity panel. Transgressive segregation was observed for γ- and α-tocochromanols in these populations, which facilitated QTL identification. These QTL and transgressive segregant families can be used in selection programs for vitamin E enhancement in maize. This work illustrates the complementary nature of biparental mapping populations and genome-wide association studies to further characterize genetic variation of tocochromanol content in maize grain.

Rice tocopherol deficiency 1 encodes a homogentisate phytyltransferase essential for tocopherol biosynthesis and plant development in rice.

RTD1 encodes a homogentisate phytyltransferase catalyzing a key step in rice tocopherol biosynthesis, confers cold tolerance and regulates rice development by affecting the accumulation of DELLA protein SLENDER RICE1. Tocopherols are one of the most important lipid-soluble antioxidants having indispensable roles in living organisms. The physiological functions of tocopherols have been comprehensively characterized in animals and artificial membranes. However, genetic and molecular functions of tocopherols in plants are less understood. This study aimed to isolate a tocopherol-deficient mutant rtd1 in rice. The rtd1 mutant showed overall growth retardation throughout the growth period. Most of the agronomic traits were impaired in rtd1. Map-based cloning revealed that the RTD1 gene encoded a homogentisate phytyltransferase, a key enzyme catalyzing the committed step in tocopherol biosynthesis. RTD1 was preferentially expressed in green leafy tissues, and the protein was located in chloroplasts. Cold tolerance was found to be reduced in rtd1. The cold-related C-repeat-binding factor (CBF)/dehydration-responsive element-binding protein 1 (DREB1) genes were significantly upregulated in rtd1 under natural growth conditions. Moreover, rtd1 exhibited a reduced response to gibberellin (GA).The transcript and protein levels of DELLA protein-coding gene SLENDER RICE 1 (SLR1) in rice was increased in rtd1. However, the GA content was not changed, suggesting a transcriptional, not posttranslational, regulation of SLR1. These findings implied that tocopherols play important roles in regulating rice growth and development.

Transient expression of the homogentisate phytyltransferase gene from Clitoria ternatea causes metabolic enhancement of α-tocopherol biosynthesis and chlorophyll degradation in tomato leaves

Homogentisate prenyltransferase (HPT) is an important enzyme involved in the α-tocopherol (vitamin E) biosynthetic pathway of all plant taxa. Tocopherol biosynthesis and chlorophyll degradation are related, but more information is needed to explain their relationship. In this study, a candidate gene for HPT from Clitoria ternatea (CtHPT) was isolated and identified via a phylogeny-based approach, and its hypothetical protein sequence was analyzed. Transient expression of CtHPT with Agrobacterium-mediated infiltration into tomato leaves was then performed and observed for the metabolic relationship between the α-tocopherol biosynthesis and chlorophyll degradation by gas chromatography–mass spectrometry. In silico analysis showed that CtHPT contained a chloroplast signal peptide and nine-transmembrane α-helixes. The results showed that, the content of α-tocopherol increased in transient expression of CtHPT, with the increased pool sizes of its biosynthetic intermediates: 2-methyl-6-phythylbenzoquinol and 2,3-dimethyl-5-phythylbenzoquinol, and the increased levels of phytol and various fatty acids. Moreover, the CtHPT transient expression was observed to cause chlorophyll deficiency in the tomato leaves with simultaneous increase of phytol and fatty acids, presumably the degradative products of chlorophyll and chloroplast membranes, respectively. It was concluded that the overexpression of CtHPT may enhance the metabolic flow of the α-tocopherol biosynthetic pathway, causing the degradation of chlorophylls, thereby increasing the supply of the precursor phytol for the α-tocopherol biosynthetic pathway.

SGD1, a key enzyme in tocopherol biosynthesis, is essential for plant development and cold tolerance in rice

Tocopherols, a group of Vitamin E compounds, are essential components of the human diet. In contrast to well documented roles in animals, the functions of tocopherols in plants are less understood. In this study, we characterized two allelic rice dwarf mutant lines designated sgd1-1 and sgd1-2 (small grain and dwarf1). Histological observations showed that the dwarf phenotypes were mainly due to cell elongation defects. A map-based cloning strategy and subsequent complementation test showed that SGD1 encodes homogentisate phytyltransferase (HPT), a key enzyme in tocopherol biosynthesis. Mutation of SGD1 resulted in tocopherol deficiency in both sgd1mutants. No oxidant damage was detected in the sgd1 mutants. Further analysis showed that sgd1-2 was hypersensitive to cold stress. Our results indicate that SGD1 is essential for plant development and cold tolerance in rice.