Highly effective mlo-based powdery mildew resistance in hexaploid wheat without pleiotropic effects.

Application of the mlo-based resistance in barley against powdery mildew attacks is a major success in crop breeding, since it confers durable disease resistance. Resistance caused by mutations in the Mlo gene seems to be ubiquitous across a range of species. This work addresses the introduction of mlo-based resistance into hexaploid wheat, which is complicated by the occurrence of three homoeologous genes: Mlo-A1, Mlo-B1 and Mlo-D1. EMS-generated mutant plants were screened for mutations in the three homoeologues. We selected and combined 6, 8, and 4 mutations, respectively, to obtain triple homozygous mlo mutant lines. Twenty-four mutant lines showed highly effective resistance towards attack by the powdery mildew pathogen under field conditions. All 18 mutations appeared to contribute to resistance; however, they had different effects on the occurrence of symptoms such as chlorotic and necrotic spots, which are pleiotropic to the mlo-based powdery mildew resistance. We conclude that to obtain highly effective powdery mildew resistance in wheat and to avoid detrimental pleiotropic effects, all three Mlo homoeologues should be mutated; however, at least one of the mutations should be of the weaker type in order to alleviate strong pleiotropic effects from the other mutations.

Analysis of barley mutants ert-c.1 and ert-d.7 reveals two loci with additive effect on plant architecture.

MAIN CONCLUSION: Both mutant ert-c.1 and ert-d.7 carry T2-T3 translocations in the Ert-c gene. Principal coordinate analyses revealed the translocation types and translocation breakpoints. Mutant ert-d.7 is an Ert-c Ert-d double mutant. Mutations in the Ert-c and Ert-d loci are among the most common barley mutations affecting plant architecture. The mutants have various degrees of erect and compact spikes, often accompanied with short and stiff culms. In the current study, complementation tests, linkage mapping, principal coordinate analyses and fine mapping were conducted. We conclude that the original ert-d.7 mutant does not only carry an ert-d mutation but also an ert-c mutation. Combined, mutations in Ert-c and Ert-d cause a pyramid-dense spike phenotype, whereas mutations in only Ert-c or Ert-d give a pyramid and dense phenotype, respectively. Associations between the Ert-c gene and T2-T3 translocations were detected in both mutant ert-c.1 and ert-d.7. Different genetic association patterns indicate different translocation breakpoints in these two mutants. Principal coordinate analysis based on genetic distance and screening of recombinants from all four ends of polymorphic regions was an efficient way to narrow down the region of interest in translocation-involved populations. The Ert-c gene was mapped to the marker interval of 2_0801to1_0224 on 3HL near the centromere. The results illuminate a complex connection between two single genes having additive effects on barley spike architecture and will facilitate the identification of the Ert-c and Ert-d genes.

Stepwise mass spectrometry-based approach for confirming the presence of benzoxazinoids in herbs and vegetables.

INTRODUCTION: Benzoxazinoids (BXs) are plant phytochemicals that have both defensive properties in plants and therapeutic effects in humans. The presence of BXs has been largely studied in the Poaceae family (monocots). To study the presence or absence of BXs in dicotyledons and monocotyledons outside the Poaceae family, parts of 24 plant species at several growth stages were selected for analysis, some of which were already known to contain BXs. OBJECTIVES: To devise a stepwise mass spectrometry-based approach for confirming the presence of BXs in plant samples, and to use the method to explore the status of BXs in selected plant species. EXPERIMENTAL: Plant samples were extracted using accelerated solvent extraction and analysed using triple-quadrupole liquid chromatography-mass spectrometry. RESULTS: The use of different columns, double mass transitions, and ion ratios proved to be a robust tool for confirming the presence of BXs in different plant species. By this method, the presence of BXs was confirmed in three of the 24 species. Double-hexose forms of BXs, which have not been reported before in dicotyledons, were confirmed to be present in the dicotyledon plants Acanthus mollis and Lamium galeobdolon, and the presence of BXs in the seeds of Consolida orientalis is reported for the first time here. High concentrations of BXs were found in the aerial parts of Acanthus mollis and Lamium galeobdolon, at 20 and 32 µmol/g plant dry weight, respectively. CONCLUSIONS: The stepwise approach described in this work confirmed the presence of BXs in new samples.

Induced Genetic Variation in Crop Plants by Random or Targeted Mutagenesis: Convergence and Differences.

New Breeding Techniques (NBTs) include several new technologies for introduction of new variation into crop plants for plant breeding, in particular the methods that aim to make targeted mutagenesis at specific sites in the plant genome (NBT mutagenesis). However, following that the French highest legislative body for administrative justice, the Conseil d'État, has sought advice from The Court of Justice of the European Union (CJEU) in interpreting the scope of the genetically modified organisms (GMO) Directive, CJEU in a decision from 2018, stated that organisms modified by these new techniques are not exempted from the current EU GMO legislation. The decision was based in a context of conventional plant breeding using mutagenesis of crop plants by physical or chemical treatments. These plants are explicitly exempted from the EU GMO legislation, based on the long-termed use of mutagenesis. Following its decision, the EU Court considers that the NBTs operate "at a rate out of all proportion to those resulting from the application of conventional methods of mutagenesis." In this paper, we argue that in fact this is not the case anymore; instead, a convergence has taken place between conventional mutagenesis and NBTs, in particular due to the possibilities of TILLING methods that allow the fast detection of mutations in any gene of a genome. Thus, by both strategies mutations in any gene across the genome can be obtained at a rather high speed. However, the differences between the strategies are 1) the precision of the exact site of mutation in a target gene, and 2) the number of off-target mutations affecting other genes than the target gene. Both aspects favour the NBT methods, which provide more precision and fewer off-target mutations. This is in stark contrast to the different status of the two technologies with respect to EU GMO legislation. In the future, this situation is not sustainable for the European plant breeding industry, since it is expected that restrictions on the use of NBTs will be weaker outside Europe. This calls for reconsiderations of the EU legislation of plants generated via NBT mutagenesis.

Development of mlo-based resistance in tetraploid wheat against wheat powdery mildew.

Powdery mildew is a severe disease in wheat. In barley, durable resistance exists, based on non-functionality of the Mlo gene. As a model to analyse the effects of mutagenesis in the homoeologous Mlo genes of wheat, we developed mlo-based powdery mildew resistance in tetraploid durum wheat. To obtain Mlo mutations, we screened a TILLING population developed in tetraploid wheat "Kronos" for which the captured exome sequence of  > 1500 lines is available. This resulted in 23 mutants for Mlo-A1 and 26 non-redundant mutants for Mlo-B1. Two Mlo-A1 and four Mlo-B1 mutants were crossed to obtain eight F2 mutant lines that showed a range of phenotypes from susceptibility to full resistance. Pot experiments under semi-field conditions confirmed the resistance levels for six of the mutants without any signs of adverse pleiotropic effects. Resistance ranking was similar across six powdery mildew isolates, indicating no isolate specificity of the mlo-based resistance. The effect of mutations in the Mlo-B1 gene was stronger than in the Mlo-A1 gene, probably reflecting differences in wild-type Mlo gene expression levels. Strong partial resistance effects were observed with single mlo-B1 mutations hence, revealing a dosage effect of mlo mutant alleles. Two of the four mlo-B1 mutations (W163* and P335L) were very strong; however, the highest combined effect was observed with the MloA-P335S/MloB-P335L combination, suggesting that non-functional, but full-length Mlo proteins might have the strongest effect compared with nonsense mutations. Our results show that mlo-based resistance might offer possibilities to introduce durable protection in tetraploid wheat against powdery mildew.

Polyamines - A New Metabolic Switch: Crosstalk With Networks Involving Senescence, Crop Improvement, and Mammalian Cancer Therapy.

Polyamines (PAs) are low molecular weight organic cations comprising biogenic amines that play multiple roles in plant growth and senescence. PA metabolism was found to play a central role in metabolic and genetic reprogramming during dark-induced barley leaf senescence (DILS). Robust PA catabolism can impact the rate of senescence progression in plants. We opine that deciphering senescence-dependent polyamine-mediated multidirectional metabolic crosstalks is important to understand regulation and involvement of PAs in plant death and re-mobilization of nutrients during senescence. This will involve optimizing the use of PA biosynthesis inhibitors, robust transgenic approaches to modulate PA biosynthetic and catabolic genes, and developing novel germplasm enriched in pro- and anti-senescence traits to ensure sustained crop productivity. PA-mediated delay of senescence can extend the photosynthesis capacity, thereby increasing grain starch content in malting grains such as barley. On the other hand, accelerating the onset of senescence can lead to increases in mineral and nitrogen content in grains for animal feed. Unraveling the "polyamine metabolic switch" and delineating the roles of PAs in senescence should further our knowledge about autophagy mechanisms involved in plant senescence as well as mammalian systems. It is noteworthy that inhibitors of PA biosynthesis block cell viability in animal model systems (cell tumor lines) to control some cancers, in this instance, proliferative cancer cells were led toward cell death. Likewise, PA conjugates work as signal carriers for slow release of regulatory molecule nitric oxide in the targeted cells. Taken together, these and other outcomes provide examples for developing novel therapeutics for human health wellness as well as developing plant resistance/tolerance to stress stimuli.

Expression analysis of the polyphenol oxidase gene in response to signaling molecules, herbivory and wounding in antisense transgenic tobacco plants.

KEY MESSAGE: We provide evidence that the expression of the PPO gene was significantly reduced in response to wounding, MeJ and herbivory in transgenic tobacco under wound-inducible OsRGLP2 promoter in an anti-sense orientation. ABSTRACT: Polyphenol oxidase (PPO) genes play an important role in plant defense mechanisms against biotic and abiotic stresses. In the present study, a 655 bp core sequence of the potato PPO gene was placed under the control of wound-inducible OsRGLP2 promoter in an anti-sense direction to evaluate its potential effects during biotic (Trialeurodes vaporariorum's infestation) and various abiotic (wounding, MeJ, ABA) stresses. Transcriptional profiling of PPO gene by real-time PCR (qRT-PCR) in transgenic tobacco revealed a significant suppression (3.5-fold) of PPO in response to wounding than control plants after 24 h. In response to MeJ at different concentrations (100 µM and 200 µM), the PPO expression was greatly down-regulated by 4.7-fold after 6 h at 100 µM MeJ, and a non-significant expression was observed with ABA treatment. Moreover, significant levels of PPO reduction (sixfolds) was found in whitefly feeding assay indicating that expression of potato PPO in an anti-sense orientation had down-regulated the PPO activity. This down-regulation of PPO by wounding, MeJ and whitefly infestation clearly links the specific expression of PPO in biotic and abiotic stresses. In the future, PPO gene suppression in transgenic plants using anti-sense potato PPO gene construct can be used to inhibit enzymatic browning in fruits and vegetables, e.g., potato.

De novo assembly of Agave sisalana transcriptome in response to drought stress provides insight into the tolerance mechanisms.

Agave, monocotyledonous succulent plants, is endemic to arid regions of North America, exhibiting exceptional tolerance to their xeric environments. They employ various strategies to overcome environmental constraints, such as crassulacean acid metabolism, wax depositions, and protective leaf morphology. Genomic resources of Agave species have received little attention irrespective of their cultural, economic and ecological importance, which so far prevented the understanding of the molecular bases underlying their adaptations to the arid environment. In this study, we aimed to elucidate molecular mechanism(s) using transcriptome sequencing of A. sisalana. A de novo approach was applied to assemble paired-end reads. The expression study unveiled 3,095 differentially expressed unigenes between well-irrigated and drought-stressed leaf samples. Gene ontology and KEGG analysis specified a significant number of abiotic stress responsive genes and pathways involved in processes like hormonal responses, antioxidant activity, response to stress stimuli, wax biosynthesis, and ROS metabolism. We also identified transcripts belonging to several families harboring important drought-responsive genes. Our study provides the first insight into the genomic structure of A. sisalana underlying adaptations to drought stress, thus providing diverse genetic resources for drought tolerance breeding research.

Genome wide characterization of barley NAC transcription factors enables the identification of grain-specific transcription factors exclusive for the Poaceae family of monocotyledonous plants.

The plant NAC transcription factors depict one of the largest plant transcription factor families. They regulate a wide range of different developmental processes and most probably played an important role in the evolutionary diversification of plants. This makes comparative studies of the NAC transcription factor family between individual species and genera highly relevant and such studies have in recent years been greatly facilitated by the increasing number of fully sequenced complex plant genomes. This study combines the characterization of the NAC transcription factors in the recently sequenced genome of the cereal crop barley with expression analysis and a comprehensive phylogenetic characterization of the NAC transcription factors in other monocotyledonous plant species. Our results provide evidence for the emergence of a NAC transcription factor subclade that is exclusively expressed in the grains of the Poaceae family of grasses. These notably comprise a number of cereal crops other than barley, such as wheat, rice, maize or millet, which are all cultivated for their starchy edible grains. Apparently, the grain specific subclade emerged from a well described subgroup of NAC transcription factors associated with the senescence process. A promoter exchange subsequently resulted in grain specific expression. We propose to designate this transcription factor subclade Grain-NACs and we discuss their involvement in programmed cell death as well as their potential role in the evolution of the Poaceae grain, which doubtlessly is of central importance for human nutrition.

Biosynthesis and chemical transformation of benzoxazinoids in rye during seed germination and the identification of a rye Bx6-like gene.

Benzoxazinoids are secondary metabolites with plant defense properties and possible health-promoting effects in humans. In this study, the transcriptional activity of ScBx genes (ScBx1-ScBx5; ScBx6-like), involved in benzoxazinoid biosynthesis, was analyzed during germination and early seedling development in rye. Our results showed that ScBx genes had highest levels of expression at 24-30 h after germination, followed by a decrease at later stages. For ScBx1-ScBx5 genes expression was higher in shoots compared with root tissues and vice versa for ScBx6-like gene transcripts. Moreover, methylated forms of benzoxazinoids accumulated in roots rather than in shoots during seedling development, in particular reaching high levels of HMBOA-glc in roots. Chemical profiles of benzoxazinoid accumulation in the developing seedling reflected the combined effects of de novo biosynthesis of the compounds as well as the turnover of compounds either pre-stored in the embryo or de novo biosynthesized. Bioinformatic analysis, together with the differential distribution of ScBx6-like transcripts in root and shoot tissues, suggested the presence of a ZmBx6 homolog encoding a 2-oxoglutarate dependent dehydrogenase in rye. The ScBx6-like cDNA was expressed in E. coli for functional characterization in vitro. LC-MS/MS analysis showed that the purified enzyme was responsible for the oxidation of DIBOA-glc into TRIBOA-glc, strongly suggesting the ScBX6-like enzyme in rye to be a functional ortholog of maize ZmBX6.

Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence.

The plant-specific NAC transcription factors have attracted particular attention because of their involvement in stress responses, senescence, and nutrient remobilization. The HvNAC005 gene of barley encodes a protein belonging to subgroup NAC-a6 of the NAC family. This study shows that HvNAC005 is associated with developmental senescence. It was significantly up-regulated following ABA treatment, supported by ABA-responsive elements in its promoter, but it was not up-regulated during dark-induced senescence. The C-termini of proteins closely related to HvNAC005 showed overall high divergence but also contained conserved short motifs. A serine- and leucine-containing central motif was essential for transcriptional activity of the HvNAC005 C-terminus in yeast. Over-expression of HvNAC005 in barley resulted in a strong phenotype with delayed development combined with precocious senescence. The over-expressing plants showed up-regulation of genes involved with secondary metabolism, hormone metabolism, stress, signalling, development, and transport. Up-regulation of senescence markers and hormone metabolism and signalling genes supports a role of HvNAC005 in the cross field of different hormone and signalling pathways. Binding of HvNAC005 to promoter sequences of putative target genes containing the T[G/A]CGT core motif was shown by direct protein-DNA interactions of HvNAC005 with promoters for two of the up-regulated genes. In conclusion, HvNAC005 was shown to be a strong positive regulator of senescence and so is an obvious target for the fine-tuning of gene expression in future attempts to improve nutrient remobilization related to the senescence process in barley.

Correlation of Deoxynivalenol Accumulation in Fusarium-Infected Winter and Spring Wheat Cultivars with Secondary Metabolites at Different Growth Stages.

Fusarium infection in wheat causes Fusarium head blight, resulting in yield losses and contamination of grains with trichothecenes. Some plant secondary metabolites inhibit accumulation of trichothecenes. Eighteen Fusarium infected wheat cultivars were harvested at five time points and analyzed for the trichothecene deoxynivalenol (DON) and 38 wheat secondary metabolites (benzoxazinoids, phenolic acids, carotenoids, and flavonoids). Multivariate analysis showed that harvest time strongly impacted the content of secondary metabolites, more distinctly for winter wheat than spring wheat. The benzoxazinoid 2-ß-glucopyranoside-2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-glc), α-tocopherol, and the flavonoids homoorientin and orientin were identified as potential inhibitors of DON accumulation. Several phenolic acids, lutein and ß-carotene also affected DON accumulation, but the effect varied for the two wheat types. The results could form a basis for choosing wheat cultivars using metabolite profiling as a marker for selecting wheat cultivars with improved resistance against Fusarium head blight and accumulation of trichothecene toxins in wheat heads.

Benzoxazinoids: Cereal phytochemicals with putative therapeutic and health-protecting properties.

Benzoxazinoids (BXs) are a group of natural chemical compounds with putative pharmacological and health-protecting properties. BXs were formerly identified in and isolated from selected dicot medicinal plants and young cereal plants. Recently, BXs were found to be present in mature cereal grains and bakery products, such that knowledge about the pharmacological properties of BXs, which until now have unknowingly been consumed through the daily bread and breakfast cereals, has come into new focus. This review discusses published results from in vitro studies and a few human and animal model studies on the health effects and pharmacological responses of various BX compounds. Many of these studies have reported antimicrobial, anticancer, reproductive system stimulatory, central nervous system stimulatory, immunoregulatory, and appetite- and weight-reducing effects of BXs and/or BX derivatives. The health benefits of wholegrain intake may be associated with the solitary and/or overlapping biological effects of fibers, lignans, phenolic acids, alkylresorcinols, BXs, and other bioactive compounds. In the context of BXs as dietary ingredients, further comprehensive investigations are required to understand their biological functions, to elucidate the underlying mechanisms, to explore their potential contribution on the health effects associated with wholegrain consumption, and to examine their potential as functional food ingredients.

NAC Transcription Factors in Senescence: From Molecular Structure to Function in Crops.

Within the last decade, NAC transcription factors have been shown to play essential roles in senescence, which is the focus of this review. Transcriptome analyses associate approximately one third of Arabidopsis NAC genes and many crop NAC genes with senescence, thereby implicating NAC genes as important regulators of the senescence process. The consensus DNA binding site of the NAC domain is used to predict NAC target genes, and protein interaction sites can be predicted for the intrinsically disordered transcription regulatory domains of NAC proteins. The molecular characteristics of these domains determine the interactions in gene regulatory networks. Emerging local NAC-centered gene regulatory networks reveal complex molecular mechanisms of stress- and hormone-regulated senescence and basic physiological steps of the senescence process. For example, through molecular interactions involving the hormone abscisic acid, Arabidopsis NAP promotes chlorophyll degradation, a hallmark of senescence. Furthermore, studies of the functional rice ortholog, OsNAP, suggest that NAC genes can be targeted to obtain specific changes in lifespan control and nutrient remobilization in crop plants. This is also exemplified by the wheat NAM1 genes which promote senescence and increase grain zinc, iron, and protein content. Thus, NAC genes are promising targets for fine-tuning senescence for increased yield and quality.

Identification of predominant genes involved in regulation and execution of senescence-associated nitrogen remobilization in flag leaves of field grown barley.

The transcriptomes of senescing flag leaves collected from barley field plots with standard or high nitrogen supply were compared to identify genes specifically associated with nitrogen remobilization during leaf senescence under agronomically relevant conditions. In flag leaves collected in field plots with high nitrogen supply, the decline in chlorophyll content was delayed. By comparing changes in gene expression for the two nitrogen levels, it was possible to discriminate genes related to nitrogen remobilization during senescence and genes involved in other processes associated with the late development of leaves under field conditions. Predominant genes that were more strongly upregulated during senescence of flag leaves from plants with standard nitrogen supply included genes encoding the transcription factor HvNAC026, serine type protease SCPL51, and the autophagy factors APG7 and ATG18F. Elevated expression of these genes in senescing leaves from plants with standard nitrogen supply indicates important roles of the corresponding proteins in nitrogen remobilization. In comparison, the genes upregulated in both flag leaf samples might have roles in general senescence processes associated with late leaf development. Among these genes were the transcription factor genes HvNAC001, HvNAC005, HvNAC013, HvWRKY12 and MYB, genes encoding the papain-like cysteine peptidases HvPAP14 and HvPAP20, as well as a subtilase gene.

Members of the barley NAC transcription factor gene family show differential co-regulation with senescence-associated genes during senescence of flag leaves.

The senescence process of plants is important for the completion of their life cycle, particularly for crop plants, it is essential for efficient nutrient remobilization during seed filling. It is a highly regulated process, and in order to address the regulatory aspect, the role of genes in the NAC transcription factor family during senescence of barley flag leaves was studied. Several members of the NAC transcription factor gene family were up-regulated during senescence in a microarray experiment, together with a large range of senescence-associated genes, reflecting the coordinated activation of degradation processes in senescing barley leaf tissues. This picture was confirmed in a detailed quantitative reverse transcription-PCR (qRT-PCR) experiment, which also showed distinct gene expression patterns for different members of the NAC gene family, suggesting a group of ~15 out of the 47 studied NAC genes to be important for signalling processes and for the execution of degradation processes during leaf senescence in barley. Seven models for DNA-binding motifs for NAC transcription factors were designed based on published motifs, and available promoter sequences of barley genes were screened for the motifs. Genes up-regulated during senescence showed a significant over-representation of the motifs, suggesting regulation by the NAC transcription factors. Furthermore, co-regulation studies showed that genes possessing the motifs in the promoter in general were highly co-expressed with members of the NAC gene family. In conclusion, a list of up to 15 NAC genes from barley that are strong candidates for being regulatory factors of importance for senescence and biotic stress-related traits affecting the productivity of cereal crop plants has been generated. Furthermore, a list of 71 senescence-associated genes that are potential target genes for these NAC transcription factors is presented.

The barley HvNAC6 transcription factor affects ABA accumulation and promotes basal resistance against powdery mildew.

Barley HvNAC6 is a member of the plant-specific NAC (NAM, ATAF1,2, CUC2) transcription factor family and we have shown previously that it acts as a positive regulator of basal resistance in barley against the biotrophic pathogen Blumeria graminis f. sp. hordei (Bgh). In this study, we use a transgenic approach to constitutively silence HvNAC6 expression, using RNA interference (RNAi), to investigate the in vivo functions of HvNAC6 in basal resistance responses in barley in relation to the phytohormone ABA. The HvNAC6 RNAi plants displayed reduced HvNAC6 transcript levels and were more susceptible to Bgh than wild-type plants. Application of exogenous ABA increased basal resistance against Bgh in wild-type plants, but not in HvNAC6 RNAi plants, suggesting that ABA is a positive regulator of basal resistance which depends on HvNAC6. Silencing of HvNAC6 expression altered the light/dark rhythm of ABA levels which were, however, not influenced by Bgh inoculation. The expression of the two ABA biosynthetic genes HvNCED1 and HvNCED2 was compromised, and transcript levels of the ABA conjugating HvBG7 enzyme were elevated in the HvNAC6 RNAi lines, but this effect was not clearly associated with transgene-mediated resistance. Together, these data support a function of HvNAC6 as a regulator of ABA-mediated defence responses for maintenance of effective basal resistance against Bgh.

Comparison of the levels of bioactive benzoxazinoids in different wheat and rye fractions and the transformation of these compounds in homemade foods.

Benzoxazinoids are important phytochemicals found in wheat and rye that are associated with plant resistance against pathogens, and recent studies have emphasized the potential health-promoting role of these compounds i.e. anti-cancer, anti-allergy and anti-inflammation. Accordingly, an understanding of their distribution in seeds and the effect of different processing techniques on their transformation will be helpful in identifying the mechanisms of their production and distribution and will support the on-going efforts to utilize these compounds in health-promoting food products. The analysis of seed fractions obtained from the milling of wheat and rye showed significantly higher concentrations of these bioactive compounds in the germ than in the other fractions, i.e. the bran and endosperm. Di-hexoses of 2,4-dihydroxy-1, 4-benzoxazin-3-one (DIBOA-glc-hexose) and 2-hydroxy-1, 4-benzoxazin-3-one (HBOA-glc-hexose) were the predominant compounds found in the different wheat and rye seed fractions followed by DIBOA-glc and DIBOA. The soaking and boiling of three rye-based breakfast cereals resulted in considerable changes in the benzoxazinoid contents. The soaking of pearled rye promoted the conversion of DIBOA-glc-hexose into DIBOA-glc. When these cereals were boiled, the increase in the DIBOA-glc content was much lower than that observed for soaking. For rye flakes, the pattern of these benzoxazinoids was different from that in pearled rye seeds. A considerable amount of the benzoxazinoids was also leached into the water during soaking or boiling. This study contributes to the understanding of the underlying processes involved in the biochemical changes of benzoxazinoids and will be the basis for future studies on other food-processing techniques.

Absorption and metabolic fate of bioactive dietary benzoxazinoids in humans.

SCOPE: Benzoxazinoids, which are natural compounds recently identified in mature whole grain cereals and bakery products, have been suggested to have a range of pharmacological properties and health-protecting effects. There are no published reports concerned with the absorption and metabolism of bioactive benzoxazinoids in humans. METHODS AND RESULTS: The absorption, metabolism, and excretion of ten different dietary benzoxazinoids were examined by LC-MS/MS by analyzing plasma and urine from 20 healthy human volunteers after daily intake of 143 µmol of total benzoxazinoids from rye bread and rye buns. The results showed that 2-ß-D-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc) and its oxidized analog, 2-ß-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc), were the major circulating benzoxazinoids. After consuming a benzoxazinoid diet for 1 week, morning urine contained eight benzoxazinoids with abundant HBOA-Glc (219 nmol × µmol⁻¹ of creatinine). The sulfate and glucuronide conjugates of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) were detected in plasma and urine, indicating substantial phase II metabolism. Direct absorption of lactam glycosides, the reduction of hydroxamic acid glycosides, glucuronidation, and sulfation were the main mechanisms of the absorption and metabolism of benzoxazinoids. CONCLUSION: These results indicate that following ingestion in healthy humans, a range of unmetabolized bioactive dietary benzoxazinoids and their sulfate and glucuronide conjugates appear in circulation and urine.